Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II–III trial

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Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II–III trial John E Wagner, John S Thompson, Shelly L Carter, Nancy A Kernan, for the members of the Unrelated Donor Marrow Transplantation Trial*

Summary Background Graft-versus-host disease (GVHD) reduces the efficacy of unrelated donor bone marrow transplantation in patients with lymphohaemopoietic malignancy. A multi-centre, randomised trial was undertaken to determine the effects of ex-vivo T-cell depletion versus methotrexate and cyclosporine immunosuppression on 3-year diseasefree survival.

Lancet 2005; 366: 733–41 Published online August 3, 2005 DOI:10.1016/S0140-6736(05) 66996-6 See Comment page 692

Methods Between Mar 1, 1995, and Oct 31, 2000, 405 patients with lymphohaemopoietic malignancy, from 15 participating centres, were randomly assigned to undergo transplantation with either T-cell depleted marrow and cyclosporine A (TCD arm; n=201) or methotrexate and cyclosporine A after transplantation of T-replete marrow (M/C arm; n=204). The primary outcome was 3-year disease-free survival and was analysed by intention to treat. Findings Five patients died before transplantation. Seven in the TCD arm received T-replete grafts. Disease-free survival at 3 years was 27% (95% CI 21–33) and 34% (27–40) in recipients of TCD and M/C, respectively (p=0·16). TCD was associated with significantly more rapid neutrophil recovery (15 days vs 20 days, p0·0001), less grade III–IV acute GVHD (18% vs 37%, p0·0001), reduced grade III–IV toxicities (19% vs 29%, p=0·017), reduced duration of initial hospitalisation, but higher risk of chronic myelogenous leukaemia relapse (20% vs 7%, p=0·009) and cytomegalovirus infection (28% vs 17%, p=0·023) than was M/C. Interpretation Disease-free survival at 3 years did not differ between TCD and M/C groups. Relapse and opportunistic infection are important obstacles to successful unrelated donor bone marrow transplantation, irrespective of the method of GVHD prophylaxis used.

Introduction Allogeneic bone marrow transplantation is widely used in the treatment of various lymphohaemopoietic malignancies.1,2 To broaden the application of this procedure to individuals who do not have an HLAmatched related donor, the use of closely HLA-matched unrelated marrow donors has been investigated.3–5 As of June 31, 2004, 5 million volunteer donors typed at HLA-A, HLA-B, and HLA-DR were registered with the National Marrow Donor Program.6 Graft-versus-host disease (GVHD) is a frequent complication of allogeneic bone marrow transplantation and is associated with significant morbidity and mortality.6,7 T-cell depletion has been shown to reduce the risk of severe GVHD after bone marrow transplantation in patients with sibling donors.8–12 Additionally, Kernan and colleagues4 recorded a significantly reduced risk of acute GVHD and improved survival in recipients of T-cell depleted unrelated marrow.4 Could T-cell depletion reduce transplantrelated mortality, improve survival, and enhance healthrelated quality of life after unrelated donor bone marrow transplantation? To address this question, the National Heart, Lung, and Blood Institute sponsored this large multi-centre, randomised trial to assess a www.thelancet.com Vol 366 August 27, 2005

physical and an immunological method of T-cell depletion in recipients of marrow from unrelated donors.

*All members of the Unrelated Donor Marrow Transplantation Trial are listed at the end of the report University of Minnesota Blood and Marrow Transplantation Program, Minneapolis, MN, USA (Prof J E Wagner MD); University of Kentucky, Lexington, KY, USA (Prof J S Thompson MD); EMMES Corporation, Rockville, MD, USA (S L Carter ScD); and Memorial Sloan-Kettering Cancer Center, New York, NY, USA (N A Kernan MD) Correspondence to: John E Wagner, Mayo Mail Code 366 UMHC, University of Minnesota Medical School, Minneapolis, MN 55455, USA [email protected]

Methods Patients Unrelated donor transplant candidates diagnosed with acute myelogenous leukaemia, acute lymphoblastic leukaemia, chronic myelogenous leukaemia, biphenotypic leukaemia, juvenile myelomonocytic leukaemia, myelodysplastic syndrome, lymphoblastic lymphoma, and non-Hodgkin’s lymphoma, and who were younger than 56 years were eligible for enrolment. Multi-organ assessment was undertaken before enrolment to ascertain the following: serum creatinine normal for age or a creatinine clearance greater than 60 mL/min per 1·73 m2, aspartate aminotransferase (AST) more than three times the typical upper limits (40 IU/L), and total serum bilirubin less than 42·75 mol/L; asymptomatic cardiac status or left ventricular ejection fraction at rest more than 40% of that improved with exercise; and asymptomatic pulmonary function or carbon monoxide diffusion in the lung, forced expiratory volume in 1 s (FEV1) and diffusion capacity more than 45% of predicted (corrected for haemoglobin). 733

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Patients with active CNS or skin leukaemic involvement, HIV seropositivity, Karnofsky performance status less than 70% or Lansky less than 50%, primary myelofibrosis, or suitable related donors were ineligible. Patients with prior autologous or allogeneic stem cell transplantation or those needing mediastinal radiation were excluded, as were individuals with uncontrolled bacterial, viral, or fungal infections, and female patients who were pregnant or breastfeeding. Eligible patients were offered participation in a phase II–III open label trial to establish the effect of GVHD prevention on 3-year disease-free survival. The treatment plan was approved by the institutional review boards of the 15 participating centres, and written informed consent was obtained from all patients before the start of treatment. Between March 1, 1995, and Oct 31, 2000, 410 patients were randomly assigned either T-cell depleted marrow and cyclosporine A (TCD arm) or methotrexate and cyclosporine A after transplantation of T-replete marrow (M/C arm).

MD, USA. Methotrexate and cyclosporine A was regarded as the standard method of GVHD prophylaxis. Two methods of T-cell depletion were used: counterflow centrifugal elutriation (Beckman Corp, Palo Alto, CA, USA), a physical method of separating T cells from haemopoietic stem cells and progenitor cells; and T10B9 (MEDI-500, Medimmune Corp, Gaithersburg, MD, USA), an antibody method of targeting the  subunit of the T-cell receptor and lysing bound cells in the presence of rabbit complement.13,14 A prescribed CD3 cell dose of 5105 cells per kg was used in recipients of elutriated marrow. The graft formulation resulted in a mean of 1 log depletion for both methods. Since conditioning regimen varied by type of GVHD prophylaxis, our study assessed the treatment package. Participants in the TCD arm received additional treatment to promote engraftment. At elutriation centres, conditioning was cyclophosphamide 120 mg/kg over 2 days and total body irradiation 1320–1375 cGy over 4 days in recipients of M/C; an identical regimen was used and supplemented with antithymocyte globulin (ATG, Pharmacia, Kalamazoo, MI, USA) 60 mg/kg over 2 days in recipients of TCD. At T10B9 centres, conditioning was cyclophosphamide 120 mg/kg over 2 days and total body irradiation 1350 cGy over 5 days in recipients of M/C, and cyclophosphamide 100 mg/kg over 2 days, cytosine arabinoside 9 g/m2 over 3 days, and total body irradiation 1410 cGy over 3–4 days in recipients of TCD. Additionally, all patients received GVHD prophylaxis with cyclosporine between day 2 and day 180 at doses needed to achieve trough levels of 200–400 ng/mL. 76% (154) of patients randomly assigned to the M/C group also received methotrexate 15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11; the other patients in the M/C group received less than four doses owing to toxicity. Transplant centres prospectively completed National Marrow Donor Program data collection forms and targeted forms for secondary endpoints and supportive care. Clinical outcome data were collected from the date of transplantation to April 30, 2002, at which time the data file was closed. Median follow-up time was 4·2 years.

Donor selection

Statistical analysis

Donors were chosen on the basis of HLA-A and HLA-B serological typing and high resolution DNA (HR DNA) typing for HLA-DRB1 (original HLA). Retrospective HLA typing was undertaken at HR DNA typing for HLAA, HLA-B, and HLA-DRB1 for 291 donor-recipient pairs.

The study was designed to have 80% power to detect a 13% difference in 3-year disease-free survival with a target sample size of 410. The study had insufficient power to distinguish differences between the two methods of T-cell depletion. The study was done with a phase II–III design. The T-cell depletion methods were qualified in the initial phase (phase II) of the study. The concept behind the phase II portion of the trial was to ensure that in a multi-centre setting the level of graft failure and acute GVHD would be acceptable with these two methods of T-cell depletion. Enrolment for the full phase III trial would be terminated if graft failure and

410 randomised

203 allocated TCD 194 received TCD 9 did not receive TCD 2 died before transplantation 5 low cell volume 1 logistics of TBI 1 conditioning regimen complications

207 allocated M/C 204 received M/C 3 did not receive M/C 3 died before transplantation

203 assessed for disease-free survival

207 assessed for disease-free survival

4 died before day 14

10 died before day 14

199 assessed for neutrophil and platelet engraftment, acute GVHD, and chronic GVHD

197 assessed for neutrophil and platelet engraftment, acute GVHD, and chronic GVHD

Figure 1: Trial profile TCD=T-cell depletion; TBI=total body irradiation; GVHD=graft-versus-host disease.

Procedures Patients were randomly assigned one of two methods of GVHD prophylaxis by a computer generated permuted block randomisation design, stratified by transplant centre. Randomisation was done centrally at the Medical Coordinating Center, EMMES Corporation, Rockville, 734

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Total

Recipient age 18 years 18–34 years 35 years Recipient sex* Male Female Recipient performance status 100% 80% and 90% 70% Recipient disease/ disease stage Chronic myelogenous leukaemia: Chronic phase 1 Accelerated phase; chronic phase 1 Missing Acute myelocytic leukaemia: Complete remission 1 and 2 Third complete remission or relapse Missing Acute lymphocytic leukaemia: Complete remission 1 and 2 Third complete remission or relapse Missing Myelodysplastic syndrome Poor risk Other leukaemia Poor risk Non-Hodgkin's lymphoma Poor risk Recipient ethnic origin White Hispanic African American Asian Other (mixed or missing) Donor/recipient CMV serostatus Positive/positive Positive/negative Negative/positive Negative/negative Unknown Donor/recipient HLA match A, B and DRB1 match A mismatch B mismatch DRB1 mismatched

M/C group

TCD group

n

(%)

n

(%)

n

(%)

96 140 174

(23%) (34%) (42%)

49 72 86

(24%) (35%) (42%)

47 68 88

(23%) (34%) (43%)

223 186

(55%) (45%)

110 96

(53%) (47%)

113 90

(56%) (44%)

141 249 20

(34%) (61%) (5%)

63 135 9

(30%) (65%) (4%)

78 114 11

(38%) (56%) (5%)

160 21 1

(39%) (5%) (0%)

79 9 0

(38%) (4%) (0%)

81 12 1

(40%) (6%) (0%)

70 31 2

(17%) (8%) (0%)

38 14 2

(18%) (7%) (1%)

32 17 0

(16%) (8%) (0%)

66 21 1

(16%) (5%) (0%)

35 10 1

(17%) (5%) (1%)

31 11 0

(15%) (5%) (0%)

23

(6%)

11

(5%)

12

(6%)

11

(3%)

5

(2%)

6

(3%)

3

(1%)

3

(1%)

0

(0%)

339 22 32 6 11

(83%) (5%) (8%) (1%) (3%)

171 10 17 3 6

(83%) (5%) (8%) (1%) (3%)

168 12 15 3 5

(83%) (6%) (7%) (1%) (3%)

73 69 105 147 16

(18%) (17%) (26%) (36%) (4%)

33 39 51 75 9

(16%) (19%) (25%) (36%) (4%)

40 30 54 72 7

(20%) (15%) (27%) (35%) (3%)

298 40 36 36

(73%) (10%) (9%) (9%)

152 21 15 19

(73%) (10%) (7%) (9%)

146 19 21 17

(72%) (9%) (10%) (8%)

* Data for sex could not be supplied for one patient. CMV=cytomegalovirus.

Table 1: Patient and graft characteristics

acute GVHD exceeded pre-specified thresholds (5% and 20%, respectively) at no cost to type I error. This was an open-label study as recipients of T-cell depleted marrow had marrow processing throughout the evening and recipients of elutriated marrow received a second infusion several hours later to attain the prescribed CD3 positive lymphocyte cell dose. The primary endpoint of disease-free survival at 3 years was analysed by intention to treat and was defined as the minimum time to relapse, death, or last follow-up. For all other outcomes, the patient population available for assessment was used, defined as patients www.thelancet.com Vol 366 August 27, 2005

surviving at least 14 days post-transplant. Survival was calculated with the Kaplan-Meier estimator.15 A modified log-rank statistic was used to assess equality of diseasefree survival by treatment because of the question of non-proportional hazards within the first 6 months.16 Otherwise, the log-rank statistic was used to test homogeneity between covariates.17 Cox’s proportional hazards model with time dependent covariates was used to create models that considered several variables: total nucleated cell dose, T-cell and CD34 dose, method of T-cell depletion, demographics, donor characteristics, disease, risk status, HLA match, recipient 735

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Total

Nucleated cell dose (106/kg) CD34 cell dose (106/kg) CD3 dose (106/kg) TCRab dose (106/kg)

M/C group

TCD group

Mean (SD)

Mean

(SD)

Mean

(SD)

168·8 2·9 16·3 15·2

276·0 3·8 30·1 28·1

(134·7) (3·4) (22·0) (21·6)

64·9 2·0 2·8 2·4

(60·2) (1·8) (12·9) (12·0)

(148·0) (2·9) (22·6) (21·6)

P

0·0001 0·0001 0·0001 0·0001

Table 2: Graft characteristics

cytomegalovirus status, infection episode, and acute GVHD grade.18 Unless indicated for the specific outcomes, there was no detectable difference between the two methods of T-cell depletion. Secondary endpoints were neutrophil and platelet recovery (absolute neutrophil count 5108/L and A

Probability of disease-free survival 1·0

M/C group (n=88) TCD group (n=94)

Cumulative incidence of relapse M/C group (n=197) TCD group (n=199)

0·8

Probabilty

Probability of disease-free survival

B

M/C group (n=207) TCD group (n=203)

Cumulative incidence of relapse M/C group (n=85) TCD group (n=93)

0·6

0·4

0·2

0 Patients at risk M/C 207 82 76 68 39 28 14 8 TCD 203 80 62 51 25 16 C 1·0 Probability of disease-free survival

1 1

88 42 94 41

Probabilty

22 11

16 8

7 4

1 1

Probability of disease-free survival M/C group (n=46) TCD group (n=42) Cumulative incidence of relapse M/C group (n=44) TCD group (n=41)

Cumulative incidence of relapse M/C group (n=49) TCD group (n=47)

0·6

34 22

D

M/C group (n=53) TCD group (n=48) 0·8

38 28

0·4

0·2

0 0 Patients at risk M/C TCD

1

2

3

4

5

6

7

0

1

Years post-transplant 53 18 48 17

18 16

17 13

8 5

5 3

2

3

4

5

6

7

4 2

0 0

Years post-transplant 0 2

0 0

46 42

19 16

18 13

15 11

9 8

7 4

Figure 2: Probability of disease-free survival and cumulative incidence estimate of relapse in recipients of TCD and M/C for all patients (A) and those with chronic myelogenous leukaemia (B), acute myelogenous leukaemia (C), and acute lymphoblastic leukaemia (D)

736

platelet count 50109/L on 7 days without transfusion), acute and chronic GVHD, infectious complications, relapse, regimen-related toxicity, resource use, and health-related quality of life. Neutrophil and platelet recoveries were analysed with the cause-specific failure probability method (or cumulative incidence) where death was treated as a competing risk and censored at end of study, and the complement of the Kaplan Meier estimator was used to calculate the cumulative probability.15,19,20 Primary graft failure was defined as failure to engraft, where engraftment is defined as achieving absolute neutrophil count 5108/L for three consecutive measurements on different days. Secondary graft failure was defined as documented engraftment (as defined above) followed by either severe neutropenia (absolute neutrophil count 5108/L) or absence of donor cells in the marrow or blood as detected by a chimerism assay. Symptoms of acute GVHD were assessed every 7 days for 98 days after transplantation. A computer generated grading algorithm was used to provide a score with an independent assessment by an expert panel review. Results were concordant in 89% of patients; discrepant scores were re-reviewed.21 Cumulative incidence estimates were calculated for acute GVHD and chronic GVHD. The maximum Bearman toxicity grade was established.22 Total number and severity of infections and infectious organisms were obtained prospectively. Infections were censored at relapse or at primary or secondary graft failure if a subsequent infusion was needed. Data for resource use included number of days of hyperalimentation, intravenous antibiotics, ventilator or intensive nursing support, number of blood products transfused, and number of outpatient clinic and home care visits during a 6-month period. Time to relapse was defined as the time to the first observation of haematological or cytogenetic changes characteristic of relapse, evidence of molecular disease with treatment intervention, or any pre-emptive treatment for relapse, including premature withdrawal of immunosuppression. Relapse of acute leukaemia was diagnosed when leukaemic blasts (immature blood cells) were documented in the blood or marrow (5%) or extramedullary site; relapse of chronic myelogenous leukaemia was diagnosed when immature haemopoietic cells were recorded in the peripheral blood and supported by myeloid hyperplasia and presence of translocations of genetic material between chromosomes 9 and 22 characteristic of chronic myelogenous leukaemia in ten or more metaphases (or more than five metaphases on two separate evaluations). The distribution of prognostic factors between M/C and TCD participants were compared by the Pearson 2 statistic for categorical variables and the Wilcoxon ranksum statistic for continuous variables.23,24 Primary causes of death were compared using the 2 statistic. www.thelancet.com Vol 366 August 27, 2005

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Role of the funding source The sponsors of the study collaborated on the study design and data interpretation. The corresponding author had full access to all the data in the study. All coauthors had final responsibility for the decision to submit for publication.

Results Figure 1 shows the trial profile. Of the patients randomised, five died before transplantation and seven in the TCD arm received T-replete grafts because of inadequate marrow harvest, logistics of total body irradiation, and conditioning regimen complications. Table 1 shows baseline characteristics for participants. The median age of the recipients was 31·2 years (IQR 19·3–41·2) and the median donor age was 36 years (19–59), with 61% of donors being men. Prognostic factors were balanced between the treatment groups (table 1). However, baseline characteristics of patients between elutriation and T10B9 centres differed. At elutriation centres, patients were younger (25·9 years vs 31·7 years, p=0·0001) and more likely to be white (94% vs 78%, p0·0001), have white donors (75% vs 66%, p=0·07), and receive a lower mean total nucleated cell (TNC) dose (123·7 vs 193·1106 TNC per kg, p0·0001) than were patients at T10B9 centres. Table 2 shows graft characteristics. For all patients, disease-free survival at 3 years was 30% (95% CI 26–35) with no significant difference between treatment groups (27% [21–33] in recipients of TCD and 34% [27–40] in recipients of M/C; difference=7% [2–16], p=0·16; figure 2). Disease-free survival was 25% for TCD recipients versus 40% for M/C recipients with chronic myelogenous leukaemia (p=0·17), 29% versus 33%, respectively, for those with acute myelogenous leukaemia (p=0·79), and 28% versus 35% for those with acute lymphoblastic leukaemia (p=0·71; figure 2). Table 3 shows prognostic factors for disease-free survival. An additional model for diseasefree survival was fit excluding infection and acute GVHD as time dependent covariates. In this model, the hazard ratio for treatment effect was 1·12 (0·88–1·41, p=0·36). Similarly, survival was 36% versus 42% for those with chronic myelogenous leukaemia (p=0·70); 31% versus 35% for those with acute myelogenous leukaemia (p=0·79); and 31% versus 34% for those with acute lymphoblastic leukaemia (p=0·66). Cumulative incidence estimate of neutrophil engraftment at day 42 was 94% (91–97) with no significant difference between TCD and M/C arms (94% [91–97] vs 93% [89–96], p=0·69). Similarly, there was no significant difference in the Kaplan Meier estimate of neutrophil recovery (96% [93–99] vs 98% [96–100], respectively; figure 3). Neutrophil recovery, however, was substantially more rapid in recipients of TCD than recipients of M/C (median 15 days vs 20 days, p0·0001). While there was no significant difference in www.thelancet.com Vol 366 August 27, 2005

Disease-free survival Transplant method† Infection episode Acute GVHD Risk status Performance status Recipient CMV serostatus Recipient age Survival Transplant method Infection episode Acute GVHD Risk status Recipient age Performance status Recipient CMV serostatus Treatment-related mortality Transplant method Recipient race Recipient age Primary disease Performance status Relapse Transplant method Risk status Performance status Acute GVHD Acute GVHD Transplant method Original HLA match T cells infused per kg (106) Chronic GVHD Transplant method Recipient age CD34 infused per kg (106) Acute GVHD Neutrophil engraftment Transplant method Original HLA match Primary disease Recipient CMV serostatus

Hazard ratio

95% CI

p

Favourable factors

1·25 2·75 1·92 1·79 1·42 1·29 1·60

0·99–1·59 1·90–3·98 1·47–2·51 1·38–2·30 1·04–1·94 1·02–1·63 1·19–2·16

0·07 0·0001 0·0001 0·0001 0·03 0·03 0·002

M/C No previous infection Acute GVHD grade 0, 1,or 2 Good risk Performance status of 90 or 100 Recipient negative CMV serostatus Recipients 18 years old

1·15 2·73 2·17 1·68 1·73 1·45 1·33

0·90–1·48 1·87–4·00 1·65–2·84 1·29–2·19 1·27–2·36 1·05–2·00 1·05–1·69

0·26 0·0001 0·0001 0·0001 0·0005 0·02 0·02

M/C No previous infection Acute GVHD grade 0, 1, or 2 Good risk Recipients 18 years old Performance status of 90 or 100 Recipient negative CMV serostatus

1·25 2·35 1·95 1·83 1·82

0·88–1·78 1·43–3·85 1·28–2·98 1·21–2·75 0·98–3·34

0·21 0·0008 0·002 0·004 0·06

M/C White recipients Recipients 18 years old Diseases other than CML Performance status of 90 or 100

1·26 3·53 1·82 0·32

0·79–2·01 2·22–5·62 1·07–3·12 0·14–0·75

0·33 0·0001 0·03 0·009

M/C Good risk Performance status of 90 or 100 Acute GVHD grade 0, 1, or 2

0·52 1·58 1·01

0·33–0·82 1·06–2·35 1·00–1·02

0·005 0·03 0·02

TCD 6 of 6 original HLA match Lower T cells infused

0·78 2·21 0·89 1·67

0·52–1·17 1·37–3·56 0·81–0·98 1·09–2·56

0·24 0·001 0·02 0·02

TCD Recipients 18 years old Higher CD34 cells infused Acute GVHD grade 0, 1, or 2

0·42 1·61 1·39 1·25

0·53–0·34 1·27–2·04 1·12–1·72 1·02–1·95

0·0001 0·0001 0·002 0·03

TCD 6 of 6 original HLA match Diseases other than CML Recipient negative CMV serostatus

CMV=cytomegalovirus; CML=chronic myelogenous leukaemia. *Final model from a Cox proportional hazards regression is presented with adjusted estimates. †Treatment arm was included in every model even if non-significant.

Table 3: Risk factors associated with disease-free survival, survival, treatment-related mortality, relapse, acute GVHD, chronic GVHD, and neutrophil engraftment after unrelated donor bone marrow transplantation, by Cox proportional hazards regression*

incidence of recovery on the basis of HLA match in M/C recipients, more rapid recovery was noted in recipients of matched versus mismatched TCD marrow (median 14 days vs 18 days, p=0·0002). Table 3 shows prognostic factors for neutrophil engraftment. Cumulative incidence estimate of platelet recovery (50 000) at 1 year for all patients was 58% (53–62) at a median of 119 days with no significant difference between the TCD and M/C arms (55% [48–62] vs 60% [53–67], p=0·15). Similarly, there was no significant difference in the Kaplan Meier estimate of platelet recovery between arms (74% [65–83] vs 84% [75–92], p=0·15; figure 3). Furthermore, rate of platelet recovery did not differ (132 days vs 115 days, respectively, p=0·21). Irrespective of GVHD prophylaxis or method of T-cell depletion, larger TNC and CD34 cell doses were 737

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Cumulative incidence of outcome

Probability of outcome M/C group (n=197) TCD group (n=199)

A

M/C group (n=197) TCD group (n=199)

B

1·0

Probabilty

0·8 0·6 0·4 0·2 0 0

10

20

30

40

50

60

C

0

2

4

6

8

10

12

0

4

8

12

16

20

24

D 1·0

Probabilty

0·8 0·6 0·4 0·2

60 70 80 90 10 0 11 0

0 10 20 30 40 50

0 Years post-transplant

Years post-transplant

Figure 3: Incidence and probability of haemopoietic recovery and acute and chronic GVHD, by treatment group A: neutrophil recovery. B: platelet recovery. C: grade III–IV acute GVHD. D: chronic GVHD.

associated with faster rates of platelet recovery. Although original HLA match had no significant effect in recipients of TCD, it was associated with a rise in the proportion of patients achieving rapid platelet recovery at 1 year among M/C recipients (49% [35–63] vs 64% [56–72], p=0·046). Cumulative incidence estimate of primary graft failure at day 28 overall was 7% (4–9) with no significant difference between TCD and M/C arms (7% [4–10] vs 6% [3–9], p=0·21). Higher incidence of primary graft failure was recorded at elutriation centres, irrespective of GVHD prophylaxis (9% [3–16] vs 10% [4–15]), than at T10B9 centres (3% [0–6] vs 7% [4–11]). Incidence of secondary graft failure at day 100 was also similar between arms (4% [2–7]) overall. However, a significantly higher incidence of secondary graft failure was noted overall at elutriation centres (4% M/C [0–11] and 14% TCD [6–25], p=0·038). Cumulative incidence estimates of acute GVHD grades II–IV were 39% (33–46) and 63% (56–69) for those receiving TCD and M/C (p0·0001) and grades III–IV disease were 18% (13–24) and 37% (30–44), respectively (p0·0001; figure 3). Cumulative incidence 738

estimates of grade III–IV acute GVHD were 30% and 26% in recipients younger than 18 years and in those 18 years or older, respectively (p=0·81). The median time to first occurrence of grade II–IV acute GVHD was significantly delayed in recipients of TCD (48·5 [29–63] vs 35·0 [26–53] days, respectively p0·0001). Cumulative incidence estimates of grades III–IV acute GVHD at 100 days were reduced with T-cell depletion in both recipients of HLA-matched marrow (15% [95% CI 9–22] vs 34% [27–43] p=0·0001), and of one antigen mismatched marrow (25% [14–37] vs 45% [31–59] p=0·015). Table 3 shows prognostic factors for grades III–IV acute GVHD. Cumulative incidence estimates of chronic GVHD at 2 years were 29% (22–35) versus 34% (27–40) for TCD and M/C (p=0·28), respectively, with no significant reduction with either method of T-cell depletion (figure 3). Time to onset of chronic GVHD did not significantly differ between groups. For recipients developing chronic GVHD, the median time of occurrence was 187 days after transplantation. Table 3 shows prognostic factors for chronic GVHD. Regimen-related toxicity was monitored for 404 patients and was reported with the Bearman toxicity scale (figure 4). Recipients of TCD had significantly less regimen-related toxicity within the first 28 days (p=0·001) irrespective of method of T-cell depletion (elutriation p=0·003; T10B9 p=0·021). Recipients of M/C had more stomatitis (p0·0001) and hepatic (p=0·0003), pulmonary (p=0·012), renal (p=0·024), and CNS (p=0·076) toxicities. Furthermore, recipients of M/C had a longer mean length of initial admission (37·8 days [SD 18·7] vs 32·0 days [SD 18·6], p=0·002) than recipients of TCD. However, the number and causes of re-admissions were similar between treatment arms. The most common indication for extended hospitalisation or re-admission was infection. Although the proportion of patients with infection and the time to first infection were equivalent for the two groups, TCD recipients had a higher proportion of life-threatening and fatal infections during the first year (44% vs 32%, p=0·012). With analysis of infection types, severe cytomegalovirus infection was reported more often in recipients of TCD (28% vs 17%, p=0·023) than in recipients of M/C as was life-threatening or fatal aspergillosis (16% vs 7%, p0·01). There were more cases of Epstein-Barr virus-lymphoproliferative disease in the TCD group than in the M/C group (5% vs 1%, respectively, p=0·018). Time in hospital after transplantation was similar in both groups. Over the first 6-month period TCD recipients used hyperalimentation less frequently (p=0·002) than did M/C recipients. Requirements for ventilator support (p=0·31), intensive nursing (p=0·36), and red-cell transfusions (p=0·45) did not differ significantly between treatment groups. Overall costs were also similar (median US$203 000 [157 000–282 000] www.thelancet.com Vol 366 August 27, 2005

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from a 5/6 to a 6/6 match and 13% decreased to matches less than 5/6. Based on original typing, disease-free survival at 3 years was 33% (95% CI 28–39) for recipients with a 6/6 HLA match versus 23% (15–31) for those with a 5/6 HLA match (p=0·044). Based on retrospective HLA typing, disease-free survival was 36% (29–43) in matched patients versus 28% (20–37) in mismatched (6/6) patients (p=0·11). Whereas TCD recipients retained faster time to engraftment irrespective of HLA match, incidence of engraftment in recipients of TCD was no different between recipients of retrospectively matched versus mismatched marrow (p=0·13).

Grade 1 Grade 2 Grade 3 Grade 4

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Figure 4: Severity of regimen-related toxicities through day 28 Relative proportions of patients with regimen-related toxicities, by the Bearman Toxicity Grading Schema, in recipients of TCD and M/C (excluding grade I–II stomatitis).

for recipients of M/C and $194 000 [136 000– 283 000] for recipients of TCD). While similar between disease groups and year of transplant, costs were greater for recipients of HLA-mismatched marrow (median $222 000 [157 000–323 000] vs $187 000 [147 000–277 000] for recipients with HLA-matched marrow p=0·038) and tended to be greater for children (p=0·27). During the study 69% (281) of patients died. Despite the reduced risk of early toxicity in TCD recipients, incidence of transplant-related mortality at 3 years after transplantation was closely similar between groups (49% [95% CI 42–56] TCD vs 49% [42–55] M/C, p=0·90). Comparing TCD and M/C, principal causes of transplant-related mortality were acute GVHD (22% vs 26%), chronic GVHD (18% vs 19%), infection without GVHD (5% vs 5%), and organ toxicity (10% vs 11%). Table 3 shows prognostic factors for transplant-related mortality. Incidence of relapse was not adversely affected by TCD except in patients with chronic myelogenous leukaemia (figure 2). Among the 78 patients who relapsed, median time to relapse was 110 days (72–249) for patients with acute myelogenous leukaemia, 146 (80–259) for those with acute lymphoblastic leukaemia, and 336 (95–542) for those with chronic myelogenous leukaemia. Table 3 shows prognostic factors for relapse. High-resolution HLA typing was obtained for 291 (71%) donor-recipient pairs for HLA-A, HLA-B, and HLA-DRB1. HLA match remained balanced between treatment groups and depletion methods. The original level of HLA match was retained in 71% of donorrecipient pairs (6/6: 167 [57%]; 5/6: 41 [14%]), and while 13% decreased from a 6/6 to a 5/6 match, 3% increased www.thelancet.com Vol 366 August 27, 2005

The successful use of bone marrow transplantation has been limited by the lack of suitable HLA-matched donors, especially in certain ethnic and racial subpopulations, as well as by acute and chronic GVHD and opportunistic infection.25–33 Acute GVHD results from the activation of donor-derived T cells, which recognise host-specific alloantigens.34 The T-cell response to these antigenic differences results in the clinical syndrome of acute GVHD. If GVHD could be adequately reduced in severity or prevented, the risk of opportunistic infection and transplant-related mortality might be sufficiently reduced to improve long-term disease-free survival. The underlying rationale for this study design was that if there was less than a 13% difference in disease-free survival by treatment group, T-cell depletion would be clinically acceptable in the setting of less GVHD. This potential effect of T-cell depletion on acute GVHD and disease-free survival is lent support by a retrospective analysis of the first 462 unrelated donor-marrow transplants.4 Several important results emerge from this randomised trial comparing M/C and TCD by an immunological and a physical approach. First, there was no statistically significant difference in disease-free survival between the TCD group (by T10B9 and counterflow elutriation) and the M/C group. However, the TCD arm showed reduced incidences of early toxicity, less acute GVHD, and more rapid neutrophil recovery. Reduction in acute GVHD in recipients of TCD had no effect on transplant-related mortality. High incidence of infection suggests the importance of delayed immune recovery irrespective of incidence of GVHD or GVHD prophylaxis used. Importantly, types of infection were closely similar, but severity and frequency of cytomegalovirus and aspergillus infections were greater in TCD recipients. Improved disease-free survival is associated with younger recipient age, good risk disease, recipient negative cytomegalovirus serostatus, and better performance status, younger donor age, and HLA match. Neutrophil engraftment is clearly comparable in recipients of TCD and M/C with faster recovery in 739

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recipients of TCD. Whereas incidence and rate of recovery could indicate greater pretransplant immunosuppression and absence of methotrexate in recipients of TCD, the importance of additional chemoradiotherapy is lent support by the higher secondary failures at elutriation centres where less immunosuppression was used. As expected, HLA match, diagnosis of acute leukaemia, and recipient CMV negative serostatus were independently associated with higher incidence of neutrophil engraftment. Furthermore, there was no difference in incidence or rate of platelet recovery between arms. However, by contrast with neutrophil recovery, rate of platelet recovery was associated with TNC (total nucleated cell dose) and CD34 cell dose. T-cell depletion has been consistently associated with reduced acute GVHD. Our study lends support to these findings in unrelated recipients of bone marrow transplant irrespective of the method of T-cell depletion. However, once severe acute GVHD occurred, risk of death was similar irrespective of the GVHD prophylaxis used. Although most studies suggest that T-cell depletion is also associated with a reduced incidence of chronic GVHD, neither method used in our study resulted in a diminished risk of chronic GVHD, suggesting that T10B9 provided only modest T-cell depletion and recipients of elutriated marrow had a prescribed CD3 dose. Similar to the results of a randomised trial with tacrolimus versus cyclosporine used in combination with methotrexate, which showed a reduction in acute GVHD in recipients of tacrolimus, a reduction of acute GVHD by T-cell depletion did not improve survival. Importantly, our study does not show that the risk of relapse is significantly affected by T-cell depletion in recipients with acute leukaemia. Whereas the risk of relapse is higher after T-cell depletion in patients with chronic myelogenous leukaemia in chronic phase, risk remains lower than that reported after T-cell depletion with sibling donors, which suggests that the graft-versusleukaemia effect could be partly retained.35,36 Use of donor lymphocyte infusions remains curative in patients with chronic myelogenous leukaemia. Close monitoring is especially important if T-cell depletion is used. In summary, partial marrow T-cell depletion, as accomplished by counterflow elutriation and T10B9 antibody plus complement, does not enhance survival in patients with lymphohaemopoietic malignancy despite a reduced risk of acute GVHD and early toxicity. Selection bias was reduced using a one to one randomised treatment allocation of patients representative of the population undergoing unrelated donor marrow transplantation. Misclassification bias was minimised by use of the disease-free survival outcome. While other methods of T-cell depletion and the accompanying treatment package could have strong effects on engraftment, GVHD, transplant-related mortality, 740

relapse, and disease-free survival, this study represents a major multi-institutional trial in unrelated donor bone marrow transplantation with a mutually agreed on outcome definition, a comprehensive data collection plan, and computer validated data entry system. The outcome definitions and study plan will assist in the design of future clinical trials in unrelated donor haemopoietic stem-cell transplants, which should focus on enhancing immune recovery, reducing opportunistic infections, and estimating effect on health-related quality of life. Members of the Unrelated Donor Marrow Transplantation Trial Participating institutions and co-investigators were: University of Minnesota (elutriation centre, n=103; Daniel Weisdorf, Jo-Ann van Burik, Stella M Davies, Shawn Fuller), Memorial SloanKettering Cancer Center (T10B9 centre, n=70; Esperanza Papadopoulos, Richard O’Reilly, Nancy Collins), Medical College of Virginia (T10B9 centre, n=53; Saul Yanovich), Wake Forest University-Baptist (T10B9 centre, n=36; David Hurd), University of Nebraska (elutriation centre, n=34; Steven Pavletic, Thomas Gross, Michael Bishop), University of Utah (T10B9 centre, n=33; Finn Petersen, Patrick Beatty), Stanford University (T10B9 centre, n=25; Robert Negrin), University of Iowa (T10B9 centre, n=19; Roger Gingrich), University of South Carolina (T10B9 centre, n=13; Jean Henslee-Downey, Adrian Gee), Ohio State University (T10B9 centre, n=6; Edward Copelan), Duke University (T10B9 centre, n=6, Joanne Kurtzberg), University of Kentucky (T10B9 centre, n=5; Gordon Phillips), Medical College of Wisconsin (T10B9 centre, n=4, James Casper, Carolyn Keever-Taylor, William Drobyski, Neal Flomenberg), Western Pennsylvania Hospital (T10B9 centre, n=2; Richard Shadduck), and University of Pittsburgh, (n=1; Albert Donnenberg), National Marrow Donor Program (Craig Howe), Fred Hutchinson Cancer Research Center (Paul J Martin), The EMMES Corporation (Donald Stablein, Adam Mendizabal, Elizabeth Wagner), NHLBI (LeeAnn Jensen, Nancy Geller, Paul McCurdy). Contributors J Wagner wrote this paper with input from all other investigators. All authors participated in the design, conduct, and analysis of the trial. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgments We thank the many clinical investigators who have advanced the field; the many physicians and nurses who have diligently cared for these patients; the many search coordinators and the dedicated staff of the National Marrow Donor Program for their work; and Angela Norman for her assistance in the preparation of this manuscript. This trial was supported by a contract from the National Heart, Lung and Blood Institute (N01-HB-47095 [JEW], N01-HB-47097 [JST], N01-HB-47094 [SLC] and N01-HB-47098 [NAK]). References 1 Horowitz MM. Uses and growth of hematopoietic cell transplantation. In: Blume KG, Forman SJ, Appelbaum FR, eds. Thomas’ hematopoietic cell transplantation, 3rd edn. Oxford, UK: Blackwell Publishing, 2003; 9–15. 2 European Group for Blood and Marrow Transplantation. Transplant activity survey 2002. Paris, France. http://www. ebmt.org/4Registry/registry6.html (accessed June, 2004). 3 Hansen JA, Clift RA, Thomas ED, Buckner CD, Storb R, Giblett ER. Transplantation of marrow from an unrelated donor to a patient with acute leukemia. N Engl J Med 1980; 303: 565–67. 4 Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from unrelated donors facilitated by the National Marrow Donor Program. N Engl J Med 1993; 328: 593–602. 5 Petersdorf EW. Hematopoietic cell transplantation from unrelated donors. In: Blume KG, Forman SJ, Appelbaum FR, eds. Thomas’ hematopoietic cell transplantation. 3rd edn. Oxford, UK: Blackwell Publishing, 2003; 1132–49.

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