Ex Vivo T Cell Depleted versus Unmodified Allografts in Patients with Acute Myeloid Leukemia in First Complete Remission

Abstract

Purpose

To retrospectively compare the clinical outcomes after transplantation of T cell depleted (TCD) and unmodified allografts in patients with acute myeloid leukemia (AML) in first complete remission (CR1).

Patients and methods

Patients received TCD grafts at Memorial Sloan-Kettering Cancer Center (MSKCC, N=115) between 2001 and 2010 using the following preparative regimens: Hyperfractionated total body irradiation (HFTBI) /thiotepa /fludarabine; HFTBI /thiotepa /cyclophosphamide; i.v. busulfan/melphalan/fludarabine. T cell depletion was performed by one of two immunomagnetic CD34+ cell selection methods for peripheral blood grafts or by soybean lectin agglutination followed by sRBC-rosette depletion for bone marrow grafts. No additional graft-versus-host disease (GVHD) prophylaxis was administered. Patients received unmodified grafts at MD Anderson Cancer Center (MDACC, N=181) after conditioning with busulfan /fludarabine and GVHD prophylaxis with tacrolimus /mini-methotrexate. Patients with unrelated or HLA-mismatched donors received anti-thymocyte globulin (ATG) at both centers with some recipients of matched related donor TCD transplants also receiving ATG depending upon the preparative regimen.

Results

TCD graft recipients were more likely to be older, receive a mismatched transplant, and have peripheral blood used as the graft source. The incidences of grade 2-4 acute GVHD and chronic GVHD were significantly lower in the TCD graft group (5% vs. 18% and 13% vs. 53%). Three-year relapse-free (RFS) and overall survival (OS) rates were 58% and 57% in recipients of TCD grafts, and 60% and 66% in recipients of unmodified grafts (P=NS).

Conclusion

Survival and RFS are similar after TCD and conventional transplants from related/unrelated donors in patients with AML in CR1 but TCD significantly reduces GVHD.

Introduction

A substantial number of acute myeloid leukemia (AML) patients relapse after achieving first hematologic complete remission (CR1)(1). Allogeneic hematopoietic stem cell transplantation (SCT) is a powerful tool to reduce the risk of leukemia relapse. SCT is currently recommended for AML patients in CR1 with poor risk cytogenetics and should be considered for those with intermediate risk(2, 3). However, preparative regimen-related toxicities and graft-versus-host disease (GVHD) associated with SCT have limited its widespread use.

GVHD can be effectively prevented by ex vivo T cell depletion of the donor graft without the morbidity associated with immunosuppressive drugs(4). The early observation of immune-mediated graft rejection with the use of T cell depleted (TCD) grafts was overcome with anti-thymocyte globulin (ATG) at the expense of delayed immune reconstitution(5).

Despite the use of TCD grafts for over 3 decades, studies comparing SCT with TCD and unmodified grafts are scarce. In a retrospective study including 146 patients with diverse hematological malignancies transplanted between 1997 and 1999, survival, GVHD rates, and quality of life were found to be similar between patients who received TCD and unmodified grafts(6). In a multi-center, randomized phase II-III trial, although acute GVHD incidence was found to be lower after SCT with TCD grafts, there was no difference in survival(7). However, in both studies, T cell depletion was accomplished by a physical method or by treatment of the graft with antibodies achieving only 1 to 2 logs of depletion compared to 3 to 5 logs of depletion that is achieved with the currently available magnetic selection methods(8). To compare the efficacy of both approaches in a more homogenous patient population and with current day practices and technology, we retrospectively evaluated the outcomes of AML patients who underwent SCT with either TCD grafts at Memorial-Sloan Kettering Cancer Center (MSKCC) or unmodified grafts at The University of Texas MD Anderson Cancer Center (MDACC), while in CR1.

Patients and Methods

After approval by MSKCC and MDACC respective institutional review boards, AML patients older than 18 years, who underwent SCT between 2001 and 2010 with ablative preparative regimens while in CR1 were identified through the institutional BMT registries. At MDACC, only those who received fludarabine-busulfan conditioning were included in the study to preserve the homogeneity of the cohort. At MSKCC, all consecutively transplanted patients with AML CR1 over this time period were included in the analysis. Demographics, disease characteristics, treatment, GVHD, and survival data were retrieved from departmental databases at the respective institutions. Complete remission was defined as ≤5% blasts in bone marrow, absence of blasts in peripheral blood, platelet count ≥100K/μL, and absolute neutrophil count ≥1000/μL. Cytogenetic risk stratification considered complex cytogenetics, -5, -5q, -7, -7q, 11q23 aberrations, inv(3), t(3;3), t(6;9), t(9;22) as poor risk, and t(8;21), t(16;16), inv(16), t(15;17) as good risk(9). Donor-recipient human leukocyte antigen (HLA) matching was established by DNA sequence-specific oligonucleotide typing for HLA-A, -B, -Cw, -DQB1, and -DRB1 loci, in both institutions.

Patients at MSKCC received TCD grafts (TCD graft group, N=115) after conditioning with one of the following preparative regimens as previously reported(10-12): 1) Hyperfractionated total body irradiation (HFTBI) 1375 cGy over 4 days followed by thiotepa 5 mg/kg/day i.v. for 2 days and fludarabine 25 mg/m²/day i.v. for 5 days beginning on the first day of thiotepa (n=29); 2) HFTBI 1375 cGy over 4 days followed by thiotepa 5 mg/kg/day and cyclophosphamide 60 mg/kg/day i.v. for 2 days (n=25); or 3) busulfan 0.8 mg/kg i.v. every 6 hours for 10 doses (n=42) or 12 doses (n=19), with doses of busulfan adjusted according to pharmacokinetics, melphalan 70 mg/m²/day i.v. for 2 days and fludarabine 25 mg/m²/day i.v. for 5 days (total n=61). Six patients in the HFTBI, thiotepa, cyclophosphamide group were also enrolled on BMT CTN 0303 (13). Patients over 60 years of age and those patients with therapy related or secondary AML received the regimen without TBI. Peripheral blood grafts underwent CD34+ cell selection using the ISOLEX 300i magnetic cell selection system (Baxter, Deerfield, IL), followed by sheep red blood cell (sRBC)-rosette depletion (n=85); or CD34+ cell selection using the CliniMACS cell selection system (Miltenyi Biotech, Gladbach, Germany) alone (n=22). Bone marrow (BM) grafts were used upon donor preference and were depleted of T cells by sequential soybean lectin agglutination and sRBC-rosette depletion (n=8). All patients received equine (total 60 mg/kg) or rabbit (2.5-5 mg/kg) anti-thymocyte globulin to prevent graft rejection except for those patients receiving a transplant from a HLA-matched related donor and conditioned with HFTBI, thiotepa, fludarabine(10). No GVHD prophylaxis was administered post-transplant.

Patients at MDACC received unmodified grafts (unmodified graft group, N=181) after an ablative, reduced-toxicity preparative regimen consisting of fludarabine 40 mg/m²/day over 1 hour and busulfan 130 mg/m²/day i.v. over 3 hours for 4 days (days -6 to -3)(14). Patients with unrelated or HLA-mismatched donors received equine (total 60 mg/kg) or rabbit (total 2.5-5 mg/kg) ATG. Tacrolimus and mini-methotrexate (5 mg i.v. on days 1, 3, 6, and 11) were used for GVHD prophylaxis. Fifteen patients also received pentostatin under an investigational protocol on days 8, 15, 22, and 30 at 1 or 1.5 mg/m².

Patients were managed clinically according to MSKCC and MDACC standard guidelines including infection prophylaxis for Pneumocystis carinii, herpes viruses, and fungus. Patients received no cytomegalovirus (CMV)–specific prophylaxis and were regularly monitored for CMV reactivation by CMV pp65 antigenemia assay of peripheral blood in both institutions. Preemptive therapy was instituted in patients with documented CMV viremia. Patients in the TCD cohort were also monitored regularly for EBV reactivation in the peripheral blood by polymerase chain reaction per institutional guidelines. Patients received G-CSF beginning at day +7 after transplantation.

GVHD was diagnosed clinically, confirmed pathologically whenever possible, and classified according to standard criteria(15). GVHD diagnosed after day 100 post transplant was classified as chronic GVHD. Only patients who engrafted were evaluable for GVHD assessment. Data was updated as of July 2011.

Patients' characteristics were compared between the TCD and unmodified graft groups using chi-square test and Wilcoxon rank sum test. Overall survival (OS) and relapse-free survival (RFS) were defined as the time from SCT until death from any cause, and disease relapse or death, respectively. Non-relapse mortality (NRM) was defined as death in a patient without leukemia relapse. Univariate probabilities of OS and RFS were estimated using the Kaplan-Meier method with 95% confidence intervals (95% CI) calculated using log-transformed intervals. Predictors of OS and RFS by 3 years post-transplant were assessed in univariate and multivariate analysis using Cox's proportional hazards regression analysis. The proportionality of the hazards assumption was tested on the basis of Schoenfeld residuals and was found to be met when assessed overall and by 3 years post-transplant.

Predictors considered included: Type of graft (TCD vs. unmodified), age (older than 50 years), donor/recipient gender (female to male vs. all other), leukemia etiology (primary, secondary, therapy-related), cytogenetic risk group, donor type (HLA-matched related vs. HLA-matched unrelated, or HLA-mismatched), and stem cell source (bone marrow vs. peripheral blood). Predictors significant on univariate analysis were evaluated in multivariate analysis. Potential interaction effects by graft type were assessed by stratified analysis, and tested statistically using first degree interaction-effect terms in Cox's regression analysis. When indicated, interaction effects were adjusted for in multivariate analysis. The cumulative incidence of NRM, leukemia relapse, and GVHD was estimated based on the cumulative incidence method to account for competing risks. Leukemia relapse, death in the absence of leukemia relapse, and relapse or death in the absence of GVHD were considered competing risks for NRM, leukemia relapse, and GVHD, respectively.

Cox's regression analysis was used to compare the rate of NRM, disease relapse and GVHD according to graft type in univariate analysis. Statistical significance was determined at the 0.05 level. Statistical analysis was performed using STATA 11.0.

Results

Patient demographic and clinical characteristics are summarized in Table 1. TCD graft recipients were significantly older, less likely to have de novo AML, and more likely to have HLA-mismatched donors. Peripheral blood was almost exclusively the graft source used in the TCD graft group. Time from achievement of CR1 to transplant was longer in patients who received unmodified grafts.

Table 1. Comparison of demographic and clinical characteristics of patients who underwent transplantation with T cell depleted (TCD) or unmodified grafts.

Characteristics	TCD N=115, n (%)	Unmodified N=181 n (%)	p
Age, years*	52 (19-71)	48 (18-63)	<0.001
>50 years	66 (57)	76 (42)	0.010
Sex = Female	58 (50)	90 (50)	NS
Time CR1 to transplant, days*	83 (12-304)	97 (8-455)	0.040
Etiology			<0.001
de novo	60 (52)	144 (80)
Secondary	38 (33)	24 (13)
Therapy-related	17 (15)	13 (7)
Cytogenetic risk status			NS
Good	1 (1)	2 (1)
Intermediate	72 (63)	103 (57)
Poor	42 (37)	76 (42)
Donor type			<0.001
Matched related	56 (49)	103 (57)
Matched unrelated	32 (28)	64 (35)
Mismatch	27 (23)	14 (8)
Donor/recipient gender			NS
Match	54 (47)	89 (49)
Mismatch	61 (53)	92 (51)
Stem cell source			<0.001
Bone marrow	8 (7)	57 (32)
Peripheral blood	107 (93)	124 (68)

^*Median (range)

CR1 indicates first complete remission

Overall survival and relapse free survival

As of July 2011, the median follow-up among surviving patients was 32 (0.5-108) and 29 (2-104) months for the TCD and unmodified graft groups, respectively. Accordingly, outcomes in the TCD and unmodified groups were compared within 3 years post-transplant. A total of 43 deaths (37%) and 18 relapses (16%) occurred in the TCD graft group, and a total of 54 deaths (30%) and 42 relapses (23%) occurred in the unmodified graft group, during the study period. Comparison of outcomes within 3 years (Table 2) showed no significant differences between TCD and unmodified graft recipients in regards to OS (57% vs. 66%, p= 0.2, Figure 1), RFS (58% vs. 60%, p= 0.7) or rate of relapse (18% vs. 25%, p= 0.3).

Table 2. Comparison of outcomes in the T cell depleted (TCD) and unmodified graft groups based on univariate analysis using Cox's proportional hazards regression.

Outcome	TCD (95% CI)	Unmodified (95% CI)	p
Relapse-free survival
1 year	62% (51-70)	65% (57-72)	0.6
3 year	58% (47-67)	60% (51-67)	0.9
Overall survival
1 year	68% (58-76)	74% (66-80)	0.3
3 year	57% (47-67)	66% (58-74)	0.2
Relapse incidence
1 year	17% (11-26)	21% (15-28)	0.4
3 year	18% (12-27)	25% (19-33)	0.9
Non-relapse mortality
100 day	8% (4-15)	3% (1-7)	0.07
1 year	18% (12-27)	13% (9-19)	0.2
3 year	24% (17-34)	16% (11-23)	0.1
Acute GVHD (grade 2-4)
100 day	5% (2-11)	18% (13-24)	0.005
Chronic GVHD
3 year	13% (8-22)	53% (46-62)	<0.001

GVHD indicates graft-versus-host disease

Figure 1. Probabilities of overall (A) and relapse-free (B) survival among recipients of T cell depleted (TCD) and unmodified grafts.

Non-relapse mortality

The cumulative incidence of NRM at day 100, 1 year and 3 years were 8%, 18%, and 24% in the TCD graft group compared with 3%, 13% and 16% in the unmodified graft group. There was no significant difference in the rate of NRM between the TCD and unmodified groups (p=0.1). Causes of death in the TCD graft group included relapsed leukemia (n=17, 40%), infection (n=11, 26%), GVHD (n=5, 12%), organ toxicities (n=5, 12%), graft failure (n=2, 4%), solid malignancies (n=2, 4%), and post-transplantation lymphoproliferative disease (n=1, 2%). In the unmodified graft group, causes of death included relapsed disease (n=27, 50%), infection (n=6, 11%), GVHD (n=12, 22%), organ toxicities (n=3, 6%), graft rejection/failure (n=4, 7%), and solid malignancies (n=1, 2%). Overall, there was no significant difference in the distribution of the causes of death between the TCD and unmodified groups. Within 100 days post-transplant, 6 (5%) and 2 (1%) patients died of infections in the TCD and unmodified groups (p 0.04), respectively.

Graft-versus-host disease

The rate of grade 2-4 acute GVHD occurring within 100 days post-transplant was significantly lower in the TCD graft group compared with the unmodified graft group (5% vs. 18%, p=0.005). There was no significant difference in the rate of grade 3-4 acute GVHD between the two groups (1% vs. 3%, p=0.3). Within 3 years post-transplant, 14 and 78 patients developed chronic GVHD, of which 5 and 55 were extensive, in the TCD and unmodified graft groups, respectively. The rate of chronic GVHD was lower in the TCD graft group (13% vs. 53%, p<0.001).

Prognostic Factors

None of the prognostic factors evaluated (listed in methods section), including the type of graft, were significantly associated with OS or RFS, except for etiology of AML (Table 3). Compared with primary AML, secondary AML was associated with significantly lower OS (HR=1.7, p 0.02), but did not impact RFS (HR=1.4, p 0.1). To determine if the factors evaluated had the same impact in the TCD and unmodified groups, we performed a stratified analysis evaluating the association between the prognostic factors and outcomes separately in these two groups. This stratified analysis showed that the impact of recipient gender on OS and RFS differed according to the type of graft. Female gender was associated with significantly lower OS (HR=2.2, p 0.01) and RFS (HR=2.3, p 0.01) in the TCD group, but not in the unmodified group (HR=0.7, p 0.2; HR=0.6, p 0.07). This association was independent of the donor's gender. All other factors considered had the comparable impact on OS and RFS in the TCD and unmodified groups.

Table 3. Predictors of overall survival (OS) and relapse-free-survival (RFS) evaluated by univariate analyses.

Value	RFS, HR@ 3 years (95% CI)	OS, HR@ 3 years (95% CI)
Age
≤50 years	Reference
>50 years	1.0 (0.7-1.4)	1.1 (0.8-1.7)
Gender
Male	Reference
Female	1.1 (0.7-1.7)	1.1 (0.7-1.5)
Etiology
de novo	Reference
Secondary	1.4 (0.9-2.2)	1.7 (1.1-2.8)*
Therapy-relate	0.9 (0.4-1.7)	0.9 (0.4-2.0)
Cytogenetic risk
Good/intermediate	Reference
Poor	1.1 (0.7-1.6)	0.9 (0.6-1.4)
Donor type
Matched related	Reference
Matched unrelated	1.1 (0.8-1.7)	1.2 (0.8-1.9)
Mismatch	1.0 (0.5-1.7)	1.2 (0.7-2.2)
Stem cell source
Bone marrow	Reference
Peripheral blood	1.4 (0.8-2.5)	1.6 (0.9-2.9)
Graft type
T cell depleted	Reference
Unmodified	0.9 (0.6-1.4)	0.7 (0.5-1.1)

^*p = 0.02

On multivariate analysis, secondary AML was significantly associated with lower OS (HR=1.7, p 0.02), and female gender in the TCD group significantly associated with lower OS (HR=1.9, p 0.004) and RFS (HR=1.7, p 0.01). There was no independent effect for graft type on OS or RFS in multivariate analysis.

Subset Analysis

Since the distribution of age older than 60 years, HLA compatibility, and AML etiology differed significantly between the TCD and unmodified groups, we compared outcomes by graft type in a relatively homogeneous group that included patients younger than 60 years of age who received a graft from a 10/10 HLA compatible donor, and who did not have secondary AML. Results of this subset analysis were comparable to those obtained overall, showing no significant difference in OS (HR=0.6, p 0.1) or RFS (HR=0.8, p 0.4) in the TCD group (n=42) compared with the unmodified group (n=135). Comparable results were obtained when analysis was restricted to younger patients with primary AML and 10/10 compatible donor.

Discussion

Ex vivo T cell depletion of the graft is the most effective method to prevent GVHD after SCT. However, approaches to T-cell depletion have varied greatly in the levels of T-cell depletion achieved, and most have also incorporated post transplant immunosuppression. As a consequence, the role of T-cell depletion alone in transplantation for hematologic malignancies has not been well established and only a minority of centers in the United States use this procedure. However, significant changes have also occurred in T cell depletion technology with CD34+ selection including methods which provide a 1-2 log₁₀ greater T-cell depletion replacing monoclonal or polyclonal antibodies directed against T cell epitopes(8, 16). Significant changes have also been made to traditional conditioning regimens primarily substituting fludarabine for cyclophosphamide and the introduction of intravenous busulfan(14, 17, 18). Furthermore, advances in molecular diagnostic and supportive care have greatly reduced transplant related mortality due to viral infections.

To compare the outcomes of TCD grafts using positive CD34 selection technology to calcineurin inhibitor based GVHD prophylaxis when used with more tolerable conditioning regimens employing fludarabine +/- busalfan, we analyzed the clinical outcomes of 115 and 181 AML patients transplanted while in CR1 with TCD and unmodified grafts at MSKCC and MDACC, respectively. Survival rates were similar, albeit, with significantly lower incidences of acute and chronic GVHD in those who received TCD grafts. None of the TCD grafts were rejected with the use of ATG and ablative conditioning.

Though T cell depletion has been associated with a higher risk of relapse in patients with chronic myeloid leukemia(19-22), this has not been the case for patients with acute leukemia. Soiffer and colleagues(23) reported their results using a monoclonal antibody for T cell depletion in patients with AML undergoing transplant in first CR. The majority of these patients had adverse clinical features or poor risk cytogenetics. The 4-year estimated incidence of relapse and event free survival were 25% and 63%. Similarly, Wagner et al.(7) reported on results of a prospective randomized trial comparing T cell depletion to unmodified transplants in patients with hematologic malignancies undergoing unrelated donor transplants. In this report, there was no statistical difference between the two cohorts with respect to the incidence of relapse for patients with AML though there was a statistically lower incidence of grade II-IV GVHD. Our own results again did not demonstrate an increased risk of relapse in the recipients of TCD grafts. However, the more intense preparative regimen used in the TCD group may have offset any reduction in graft-versus-leukemia effect.

More recently, in the prospective Blood and Marrow Transplant Clinical Trials Network (BMT-CTN) 0303 trial of TCD transplants in AML(13), the relapse incidence was reported as 17% at 3 years among those in CR1, similar to those reported in prospective trials of T cell replete transplants(24-26). Furthermore, Pasquini et al. compared the outcomes of patients treated on the BMT-CTN 0303 TCD trial to similar patients who received unmodified grafts between 2003 and 2006 on BMT-CTN 0101 protocol. No significant difference in relapse incidence, NRM or survival was observed between the two patient groups(27).

In our study cohort, both acute and chronic GVHD rates were lower in the recipients of TCD grafts compared to the recipients of unmodified grafts, despite the higher prevalence of mismatched donors in the former group. Ex vivo T cell depletion of the donor graft has been consistently associated with reduced GVHD and is considered the most effective method for prevention of GVHD(7, 28, 29). However, the degree of T cell depletion and methods used may influence the incidence of GVHD(4, 28, 30-32). With 1 to 2 logs of T cell depletion, Lee et al. reported no difference in GVHD rates between TCD and T cell replete transplants from unrelated donors. With the T cell depletion techniques utilized by the MSKCC group, 3-4 logs of T cell depletion were achieved resulting in the significantly lower incidence of both acute and chronic GvHD seen in this group.

In our cohort, ex vivo T cell depletion was not associated with improved OS or RFS rates when compared to unmodified grafts, despite having lower rates of acute and chronic GVHD and similar relapse risk. OS in both groups was comparable to that previously reported in AML patients in CR1(26, 33). Likewise, previous reports comparing TCD and unmodified grafts did not demonstrate any difference in survival(6, 7) suggesting that advances in SCT affected both methods equally. Though there was a trend towards a higher NRM at day 100 in the TCD cohort, there was no difference between the two groups in 1-year and 3-year NRM. The early NRM in the TCD cohort was attributed to infectious deaths, most of which were in the early peri-transplant period and bacterial in nature. Therefore, they could not be attributed to delayed quantitative and functional recovery of T cells(5, 34-37). However, the difference in intensities of the TCD cytoreductive regimens compared to the MDACC busulfan/fludarabine regimen could potentially explain the trend towards a higher early NRM in the TCD group.

Relapse was the most important cause of treatment failure in both groups, and strategies aimed at preventing post transplant relapse will be essential to improve transplant outcomes regardless of graft source. Moving forward the ability to deliver post transplant therapies will need to be carefully assessed when comparing TCD to other GVHD prevention strategies.

Our study has several limitations, the retrospective nature of data collection being the foremost. This has led to significantly different patient/donor characteristics between the two groups. Secondly, a portion of the patients were treated under investigational trials that may have led to selection bias. Moreover, comparison of transplant procedures between the two different centers may have introduced confounding variables that we may have not accounted for, i.e. patient selection and differences in supportive therapy.

It appears from this retrospective study that survival is similar between TCD transplants and conventional transplants with a reduced GVHD rate after the former. Our results support the need for a randomized prospective trial comparing TCD and unmodified grafts in homogeneous populations.