Scholarly article on topic 'Low Risk of Chronic Graft-versus-Host Disease and Relapse Associated with T Cell–Depleted Peripheral Blood Stem Cell Transplantation for Acute Myelogenous Leukemia in First Remission: Results of the Blood and Marrow Transplant Clinical Trials Network Protocol 0303'

Low Risk of Chronic Graft-versus-Host Disease and Relapse Associated with T Cell–Depleted Peripheral Blood Stem Cell Transplantation for Acute Myelogenous Leukemia in First Remission: Results of the Blood and Marrow Transplant Clinical Trials Network Protocol 0303 Academic research paper on "Clinical medicine"

CC BY-NC-ND
0
0
Share paper
OECD Field of science
Keywords
{AML / GVHD / Prophylaxis / Transplantation}

Abstract of research paper on Clinical medicine, author of scientific article — Steven M. Devine, Shelly Carter, Robert J. Soiffer, Marcelo C. Pasquini, Parameswaran N. Hari, et al.

Graft-versus-host disease (GVHD) is most effectively prevented by ex vivo T cell depletion (TCD) of the allograft, but its role in the treatment of patients undergoing allogeneic hematopoietic cell transplantation (HCT) for acute myelogenous leukemia (AML) in complete remission (CR) remains unclear. We performed a phase 2 single-arm multicenter study to evaluate the role of TCD in AML patients in CR1 or CR2 up to age 65 years. The primary objective was to achieve a disease-free survival (DFS) rate of >75% at 6 months posttransplantation. A total of 44 patients with AML in CR1 (n = 37) or CR2 (n = 7) with a median age of 48.5 years (range, 21-59 years) received myeloablative chemotherapy and fractionated total body irradiation (1375 cGy) followed by immunomagnetically selected CD34-enriched, T cell‒depleted allografts from HLA-identical siblings. No pharmacologic GVHD prophylaxis was given. All patients engrafted. The incidence of acute GVHD grade II-IV was 22.7%, and the incidence of extensive chronic GVHD was 6.8% at 24 months. The relapse rate for patients in CR1 was 17.4% at 36 months. With a median follow-up of 34 months, DFS for all patients was 82% at 6 months, and DFS for patients in CR1 was 72.8% at 12 months and 58% at 36 months. HCT after myeloablative chemoradiotherapy can be performed in a multicenter setting using a uniform method of TCD, resulting in a low risk of extensive chronic GVHD and relapse for patients with AML in CR1.

Academic research paper on topic "Low Risk of Chronic Graft-versus-Host Disease and Relapse Associated with T Cell–Depleted Peripheral Blood Stem Cell Transplantation for Acute Myelogenous Leukemia in First Remission: Results of the Blood and Marrow Transplant Clinical Trials Network Protocol 0303"

ASBMI

American Society for Blood and Marrow Transplantation

Low Risk of Chronic Graft-versus-Host Disease and Relapse Associated with T Cell-Depleted Peripheral Blood Stem Cell Transplantation for Acute Myelogenous Leukemia in First Remission: Results of the Blood and Marrow Transplant Clinical Trials Network Protocol 0303

Steven M. Devine,1 Shelly Carter,2 Robert J. Soiffer,3 Marcelo C. Pasquini,4 Parameswaran N. Hari,4 Anthony Stein,5 Hillard M. Lazarus,6 Charles Linker,7 Edward A. Stadtmauer,8 Edwin P. Alyea, III,3 Carolyn A. Keever-Taylor,4 Richard J. O'Reilly9

Graft-versus-host disease (GVHD) is most effectively prevented by ex vivo T cell depletion (TCD) of the allograft, but its role in the treatment of patients undergoing allogeneic hematopoietic cell transplantation (HCT) for acute myelogenous leukemia (AML) in complete remission (CR) remains unclear. We performed a phase 2 single-arm multicenter study to evaluate the role of TCD in AML patients in CRI or CR2 up to age 65 years. The primary objective was to achieve a disease-free survival (DFS) rate of >75% at 6 months posttransplantation. A total of 44 patients with AML in CRI (n = 37) or CR2 (n = 7) with a median age of 48.5 years (range, 21-59 years) received myeloablative chemotherapy and fractionated total body irradiation (1375 cGy) followed by immunomagnetically selected CD34-enriched, T cell—depleted allografts from HLA-identical siblings. No pharmacologic GVHD prophylaxis was given. All patients engrafted. The incidence of acute GVHD grade II-IVwas 22.7%, and the incidence of extensive chronic GVHD was 6.8% at 24 months. The relapse rate for patients in CRI was 17.4% at 36 months. With a median follow-up of 34 months, DFS for all patients was 82% at 6 months, and DFS for patients in CRI was 72.8% at 12 months and 58% at 36 months. HCT after myeloablative chemoradiotherapy can be performed in a multicenter setting using a uniform method of TCD, resulting in a low risk of extensive chronic GVHD and relapse for patients with AML in CRI. Biol Blood Marrow Transplant 17: 1343-1351 (2011) © 2011 American Society for Blood and Marrow Transplantation

KEY WORDS: AML, GVHD, Prophylaxis, Transplantation

From the 'Medicine Department of Ohio State University, Colum-

bus, Ohio; 2EMMES Corporati on, Rockville, Maryland;

3Dana-Farber Cancer Institute, Boston, Massachusetts;

Medical College of Wisconsin, Milwaukee, Wisconsin; City

of Hope, Duarte, California; 6University Hospitals Case Medi-

cal Center, Cleveland, Ohio; 7University of California, San

Francisco, California; Abramson Cancer Center of the University of Pennsylvania, Philadelphia, Pennsylvania; and 'Memorial

Sloan-Kettering Cancer Center, New York, New York. This study was presented in part as an oral presentation at the 2009 American Society of Hematology Meeting, New Orleans, Louisiana, December 7, 2009. Financial disclosure: See Acknowledgments on page 1350. Correspondence and reprint requests: Steven M. Devine, MD, B316

Starling-Loving Hall, 320 W 10th Ave, Columbus, OH 43210 (e-mail: steven.devine@osumc.edu). Received November 4, 2010; accepted February 4, 2011 © 2011 American Society for Blood and Marrow Transplantation 1083-8791/$36.00 doi: 10.1016/j.bbmt.2011.02.002

INTRODUCTION

Allogeneic hematopoietic cell transplantation (HCT) is the most effective means for preventing relapse in patients with acute myelogenous leukemia (AML) in complete remission (CR) [1-5]. However, both acute and chronic graft-versus-host disease (GVHD) frequently occur after HCT and adversely affect quality of life and overall survival [1,6-8]. GVHD is most commonly prevented by pharmacologic therapy, but is most effectively prevented by ex vivo T cell depletion (TCD) of the allograft [9,10]. However, the use of TCD is limited by logistical difficulties, lack of a Food and Drug Administration (FDA)-approved method, and concerns regarding potential risks of graft rejection, posttransplantation infection, and leukemic relapse. Nonetheless, several recent TCD studies involving patients with AML suggest a low risk of GVHD and relapse with this approach, particularly

in patients undergoing HCT while in remission

[11-15].

The goal of the present study was to determine whether the encouraging results reported by single centers could be reproduced in a multicenter setting. We selected a TCD method that would remove almost all T cells and thus eliminate the need for posttransplantation pharmacologic GVHD prophylaxis. We limited enrollment to adult patients with AML in first CR (CR1) or second CR (CR2) and administered the TCD graft after an intensive pretransplantation conditioning regimen, reasoning that the potentially undesirable effects of TCD (eg, graft rejection, relapse) would be reduced if combined with a conditioning regimen that is highly immunosuppressive and antileu-kemic. Our results demonstrate the feasibility and effectiveness of this uniform approach.

METHODS

Patients and Donors

Patients aged 18-65 years with a diagnosis of AML in first or second complete morphological remission (CR1 or CR2) according to International Working Group criteria were eligible for this study [16,17]. Patients with acute promyelocytic leukemia and t(15;17) or core binding factor AML [M4Eo with inv16 and AML with t(8;21)] in CR1 were not eligible. Patients had to have a HLA-identical sibling donor willing to consent and able to donate a granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem/progenitor cell (PBSC) allograft and normal organ function, and had to be HIV-negative.

Sibling donors had to be younger than 75 years, weigh more than 25 kg, and be seronegative for human immunodeficiency virus, hepatitis B/C virus, human T-lymphotropic virus I/II, and syphilis. Both patients and donors provided written informed consent in accordance with the Helsinki Declaration protocol for a study approved by the institutional review board at each participating institution. The study was also approved by the FDA under an Investigational Device Exemption (BB-IDE#11965), held by the National Heart, Lung, and Blood Institute, and was monitored by an independent Data Safety and Monitoring Board appointed by the National Heart, Lung, and Blood Institute. The clinical trial is registered at Clinical Trials.gov as NCT00201240.

Conditioning Regimen

All patients received a conditioning regimen consisting of hyperfractionated total body irradiation (TBI) at a total dose of 1375 cGy given over 4 days (on days -9, -8, -7, and -6) at a dose rate of <20 cGy/ minute. Doses of 125 cGy/fraction were administered

at a minimum interval of 4 hours between fractions, 3 times daily, for a total of 11 doses. Sequential doses of TBI were administered in an anterior/posterior or lateral orientation at the discretion of the radiation oncologists at each center, but the technique was kept consistent within each institution. Compensators and lung blocks yielding a minimum lung dose of 800 cGy were allowed based on institutional practice. After TBI, thiotepa was administered at a dose of 5 mg/kg (ideal body weight)/day i.v. for 2 days on days -5 and -4, followed by cyclophosphamide 60 mg/kg (ideal or adjusted body weight)/day for 2 days (days -3 and -2). A single 2.5 mg/kg dose of rabbit anti-thymocyte globulin (Thymoglobulin; Genzyme, Cambridge, MA) was given as an i.v. infusion over 6-8 hours on day -4.

Donor Mobilization and TCD Procedures

Donors received at least 5 consecutive doses of G-CSF 10-16 mg/kg once daily by s.c. injection. Leu-kapheresis was initiated on day 5 of G-CSF therapy. To maximize the CD341 cell dose transplanted, 2 consecutive days of leukapheresis (days 5 and 6) were planned for all donors. A third day of leukapheresis was allowed if necessary to ensure infusion of a minimum CD341 cell dose (see the next section).

CD341 cell selection with the Miltenyi Clini-MACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was the sole method of TCD used in this clinical trial. TCD was performed in accordance with procedures outlined in the CliniMACS user's operating manual, as well as institutional standard operating procedures validated at each study site. Leukapheresis products were either processed on the same day as collected or refrigerated overnight (at 1-8°C) and processed the next morning.

Target Allograft Composition

The target allograft cell composition following TCD was a CD341 cell dose of $5.0 x 106/kgrecipient weight in combination with a CD31 cell dose <1.0 x 105/kg recipient weight. A CD341 cell dose of 1.0 x 106/kg was the absolute minimum at which a recipient could still be considered evaluable for study endpoints. If this minimal dose was not achieved after 2 days of leukapheresis, then donors underwent a third collection. In that case, cells from the third collection were not to be CD34+-selected. CD34+-selected allografts were transplanted in accordance with institutional protocols on day 0 and sometimes also on day 11 post-HCT.

Supportive Care and Patient Assessment

The protocol specified that no posttransplantation pharmacologic GVHD prophylaxis would be given as long as the CD31 cell dose transplanted was <1.0 x 105/kg. Hematopoietic growth factors were not

routinely administered after HCT. Blood cytomegalo-virus (CMV) surveillance was performed weekly with either quantitative polymerase chain reaction or anti-genemia assay through day 1100, and preemptive treatment was initiated as required according to institutional guidelines. Surveillance for Epstein-Barr virus (EBV) infection was performed weekly using a realtime quantitative EBV DNA polymerase chain reaction plasma-based assay through day +100 and then monthly up to day +180. EBV assays were sent from each center to a centralized laboratory at the University of Washington [18]. Patients with a blood EBV DNA concentration of >1000 copies/mL on any test were scheduled to receive preemptive rituximab at 375 mg/m2 for at least 1 dose; rituximab was continued weekly if the concentration remained >1000 copies/ mL after the first dose.

Acute and chronic GVHD were graded according to established criteria [19,20]. Patients were considered evaluable for GVHD if they engrafted. Organ toxicity was graded according to the National Cancer Institute's Common Terminology Criteria for Adverse Events version 3.0.

Donor hematopoietic chimerism was assessed on samples of unmanipulated bone marrow as well as on separated T cells (CD3+) from peripheral blood obtained on days +28, +100, +180, and +365 using standard methods at each institution. Peripheral blood samples also were obtained for standard lymphocyte immunophenotyping at the same time points. No donor leukocyte infusions or other interventions were planned based on the results ofthe chimerism analyses.

Definitions

Neutrophil engraftment was defined as an increase in absolute neutrophil count to $ 500/mL for 3 consecutive days following a conditioning regimen-induced nadir. Platelet engraftment was defined as the first day of 3 consecutive platelet count measurements >50,000/mL without the aid of transfusion for 7 consecutive days. Primary graft failure was defined as failure of neutrophil engraftment by day +30. Secondary graft failure was defined as primary engraftment followed by a subsequent decline in the absolute neutro-phil count to <500/mL with no apparent cause, such as drugs or opportunistic infection, and unresponsive to hematopoietic growth factor therapy.

The Southwest Oncology Group/Eastern Cooperative Oncology Group classification system was used to categorize the risk of relapse in patients with AML in CR1 based on cytogenetic findings [4]. Abnormalities not included in the Southwest Oncology Group/Eastern Cooperative Oncology Group definition were categorized according to the Cancer and Leukemia Group B classification system [21]. Patients with a normal karyo-type at diagnosis found to have a Flt3 internal tandem

duplication mutation were placed in the intermediate-risk cytogenetic category but were categorized as being at high risk for relapse based on previous studies [22,23].

Statistical Considerations

This study was a phase II single-arm multicenter trial with a primary objective of determining the disease-free survival (DFS) probability at 6 months post-HCT. This early DFS time point was chosen to begin planning for a follow-up trial to compare this approach with other GVHD prophylaxis strategies if the results appeared promising. DFS was defined as the minimum time from HCT to relapse or death, with censoring if the patient was alive and disease-free by May 10, 2010. The anticipated 6-month DFS probability was 75%, and the lower boundary for proceeding to a phase III trial was a DFS probability of >0.55. The primary hypothesis can be described as Ho: P #.55 versus Hj: P >.55. When the true DFS percentage is 75% with a sample size of 45, there is 84% power at a= .05 to rule out a DFS percentage of <55%. Overall survival (OS) was defined as the time from HCT to death, with censoring if the patient was alive at the data cutoff date.

Kaplan-Meier estimates were computed for DFS and OS, with Greenwood's formula as the variance estimate [24]. Cumulative incidence curves incorporating death as a competing risk were computed for time to neutrophil and platelet engraftment, relapse, and acute and chronic GVHD [25]. The cumulative incidence of TRM was calculated with relapse as a competing risk. All calculations were performed using SAS version 9.1.2 (SAS Institute, Cary, NC).

RESULTS

Patient Characteristics

Patient and donor characteristics are presented in Table 1. A total of 47 patients were enrolled at 8 centers between October 2005 and December 2008. Three of these patients were enrolled but did not proceed to transplantation on this study due to withdrawal of informed consent (n = 2) or disease progression before the onset of conditioning (n = 1); these 3 patients are not discussed further in this report. A total of 44 patients underwent HCT in this study.

Apheresis Products and Graft Processing

A total of 84 products were processed for the 44 patients who underwent HCT. Products from multiple days of collection were pooled for CD34-enrichment in 2 cases, and products from a single collection were split for enrichment on 2 columns in 4 cases. The results of the CD34+ cell selection processing are presented in Table 2. All patients received >2.0 x 106

Table 1. Recipient and Donor Characteristics

Age, years, median (range) 48.5 (2I-59)

Patient age by decade, n

20-29 7

30-39 4

40-49 I4

50-59 I9

Sex, n

Male I6

Female 28

Performance status (KPS), n

100% I7

90% I7

Leukemia stage, n

CRi 37

Race, n

White 42

Other 2

Cytogenetic risk (CRI/CR2), n

Favorable 0/I

Intermediate 26/2

Unfavorable II/3

Unknown 0/I

Recipient CMV serostatus I7/27

positive/negative, n

CD341 cells/kg, and 86% received >5.0 x 106/kg. No patient received more than the targeted CD31 cell dose of 1.0 x 105/kg.

slow, with median levels <200/mL until 1 year post-HCT.

Acute and Chronic GVHD

All patients were evaluable for acute GVHD. Ten patients developed grade II (n = 8) or III (n = 2) acute GVHD, and 10 patients developed grade I acute GVHD. No patient developed grade IVacute GVHD. Of the patients with grade II or III acute GVHD, 7 had gastrointestinal (GI) involvement (including 3 with upper GI involvement only), 6 had cutaneous involvement, and 2 had liver involvement. The median time to onset of any acute GVHD was 23 days (range, 14100 days). The cumulative incidence of acute GVHD grade II-IVat 100 days was 22.7% (95% confidence interval [CI], 10.2%-35.3%) (Figure 2A). The cumulative incidence of acute GVHD grade III-IV at 100 days was 4.5% (95% CI, 0-10.8%) (Figure 2B). Eight patients developed chronic GVHD, at a median of 129 days post-HCT (range, 109-391 days). Chronic GVHD was limited in 5 patients and extensive in 3 patients. The cumulative incidence of any chronic GVHD was 19.0% at 24 months (95% CI, 6.8%-31.1%) (Figure 2C), and the cumulative incidence of extensive chronic GVHD was 6.8% (95% CI, 0-14.4%) at 24 months.

Engraftment and Chimerism

All patients engrafted neutrophils and platelets, at a median of 12 days (range, 9-19 days) and 16 days (range, 13-159 days) posttransplantation, respectively. There were no primary graft failures. One female patient who underwent HCT while in CR1 using a male donor developed secondary graft failure on day + 54 and later died of fungal pneumonia. No patient received a donor leukocyte infusion. Donor chimerism values are given in Table 3. The degree of donor chimerism was not predictive of relapse.

Immune Reconstitution

Quantitative recovery of lymphocytes is depicted in Figure 1. CD41 and CD81 reconstitution was

Table 2. Results of CD34 Enrichment Procedures

TNC/product CD34 selected

TNC processed/patient

Total CD34 dose processed/patient

Final CD34 dose/kg transplanted

CD34 yield (%)/product CD34 selected

CD34 purity (%)/product CD34 selected

Total CD3 dose processed/patient

Final CD3 dose/kg transplanted

Logio T cell depletion/product CD34 selected

7.2 x I010 (2.1-15.5) 13.4 x I010 (5.4-26.9) 10.0 x I08 (2.9-34.6) 7.9 x I06 (2.4-31.3) 65.5% (27.2-125.5) 96.7% (61.5-99.8) 3.2 x I010 (1.3-8.5) 6.6 x I03 (I.I-84.9) 4.9 (3.2-5.9)

Data are median and range. The total number of products CD34 selected was 84.

Opportunistic Infections

Thirty-four of the 44 patients (77%) experienced at least 1 documented infection. Bacterial infections were observed in 27 patients (61%); invasive fungal infections in 5 patients (11%); and viral infections (CMV or EBV) in 25 patients (57%). CMV viremia requiring preemptive treatment was observed in 11 patients (32%). Twenty-one patients were considered at high risk for CMV reactivation due to being CMV-seropositive (n = 17) or, if CMV-seronegative, having a seropositive donor (n = 4). Nine of these 21 patients (43%) developed CMV viremia. Eight patients (18%) developed EBV viremia (>1000 copies/mL), at a median of 114 days (range, 34-349 days) post-HCT, requiring at least 1 dose of rituximab (one 375-mg/m2 dose in 7 patients and 4 doses in 1 patient). One patient developed posttransplantation lymphoproliferative disorder (PTLD) with a large-cell lymphoma involving the liver and died secondary to progressive donor cell-derived EBV-related PTLD on day +75. The cumulative incidence of EBV viremia at 12 months was 18.2% (95% CI, 6.6%-29.7%).

Nonhematologic Toxicity

The most common grade 3-5 nonhematologic tox-icities related to the conditioning regimen were GI (mucositis/stomatitis) and pulmonary (Table 3).

Table 3. Results of Transplantation

Hematopoietic engraftment, days, median (range)

Neutrophils 12 9-19)

Platelets 16 13-159)

Hematopoietic chimerism, donor %, median range)

T cell

Day +28 95 0-100)

Day + 100 71 0-100)

Day + 180 80 11-100)

Day +365 95 0-100)

Myeloid

Day +28 100 75-100)

Day + 100 100 69-100)

Day + 180 99 8-100)

Day +365 100 80-100)

Nonhematologic toxicity, % (grade) or number

Mucositis/stomatitis 34% 3)

Pulmonary 7% 3/4)

Hepatic veno-occlusive disease 5% mild)

2% moderate)

Thrombotic microangiopathy 2% 4)

Hemodialysis n 5 2

Seizures n 5 1

Acute GVHD (grade), cumulative incidence, % (95% CI)

II-IV 23 10-35)

III-IV 5 0-11)

Chronic GVHD at 24 months, cumulative

incidence, % 95% CI)

Limited/extensive 19 7-31)

Extensive only 7 0-14)

Relapse, cumulative incidence, % 95% CI)

All patients

12 months 21 8-33)

36 months 24 11-37)

CR1 only

12 months 14 2-25)

36 months 17 4-30)

CR2 only

12 months 57 15-99)

36 months 57 15-99)

TRM, cumulative incidence, % (95% CI)

12 months 14 3-24)

36 months 23 9-37)

DFS, Kaplan-Meier estimate, % 95% CI)

All patients

6 months 82 70-93)

12 months 66 52-80)

36 months 53 37-69)

CR1 only

12 months 73 58-87)

36 months 58 40-76)

CR2 only

12 months 29 0-68)

36 months 29 0-68)

OS, Kaplan-Meier estimate, % 95% CI)

All patients

12 months 77 65-90)

36 months 56 40-73)

CR1 only

12 months 81 68-93)

36 months 60 42-78)

CR2 only

12 months 57 17-98)

36 months 38 0-84)

Transplantation-Related Mortality

Nine patients died from causes other than relapse, at a median of 188 days (range, 60-762 days) post-HCT. The cumulative incidence of transplantation-related mortality (TRM) was 14% (95% CI,

3.4%-24%) at 12 months, 20% (95% CI: 7.1-32.7) at 24 months, and 23.2% (95% CI: 9.3-37.1) at 36 months (Table 3).

Relapse, DFS, and OS

The median follow-up for all surviving patients was 34 months (range, 11.5-51.5 months). Ten patients relapsed (6 who underwent HCT in CR1 and 4 who underwent HCT in CR2), at a median of 198 days post-HCT (range, 74-664 days). The cumulative incidence of relapse for the entire group was 20.6% (95% CI, 8.4%-32.8%) at 12 months and 23.7% (95% CI, 10.5%-37%) at 36 months (Figure 3). Of the 10 patients who relapsed, 4 had intermediate-risk cytogenetics, 5 had high-risk cytogenics, and 1 had unknown cytogenics. The DFS rate at 6 months post-HCT was 81.8% (95% CI, 70.3%-93.4%) for the entire group. DFS and OS by stage are given in Tables 3 and Figure 4. The most common cause ofdeath was relapse (Table 3).

DISCUSSION

These results demonstrate for the first time in a prospective multicenter setting that a strategy combining intensified myeloablative conditioning followed by transplantation of CD34-enriched, T cell-depleted PBSCs from HLA-identical sibling donors can result in consistent engraftment with a low risk of severe acute and chronic GVHD (<10% for both) and a low risk of relapse in patients with AML in CR1.

We chose a method for TCD that uses immunomag-netic selection of CD34-expressing cells based on previous studies that suggested reliable CD34 yields, high CD34 purity, and vigorous TCD [11,12,26,27]. The device (Miltenyi CliniMACS) was made available through an Investigational Device Exemption filed with the FDA. We have demonstrated that this method is highly reproducible from center to center, resulting in a consistently high degree of CD34 purification, TCD, and sterility. The CD34-enrichment procedure resulted in a median CD3 depletion of 4.9 logs. In all recipients, the CD3 dose was below the protocol-specified limit of 1 x 105/kg. The resulting low risk of grade II-IV and particularly grade III-IV acute GVHD despite the absence of pharmacologic GVHD prophylaxis suggests that this threshold T cell dose is appropriate in the HLA-matched sibling setting [28]. Although about 40% of patients still developed some acute GVHD, 13 of the 20 patients with acute GVHD had only cutaneous involvement which was manageable with corticosteroids. Further study is needed to determine whether this same threshold CD3 dose can be used in recipients of CD34-enriched unrelated donor PBSC grafts.

The median CD34 dose transplanted in this study was 7.9 x 106/kg, and, notably, 86% of the recipients

Figure 1. Recovery of lymphocytes by day posttransplantation. Values are expressed as median ± standard error. Shaded areas represent the 25th-75th percentile range for normal individuals. (A) CD41 cells. (B) CD81 cells. (C) NK cells (CD561 cells). (D) B cells.

were able to achieve the target CD34 dose of 5.0 x 106/kg. This resulted in a primary engraftment rate of 100%. One patient experienced secondary graft failure despite receiving an initial CD34 dose of 10.1 x 106 CD341 cells/kg. The pathophysiology of secondary graft failure is complex, and multiple factors may contribute to this event. Overall, the risk of primary or secondary graft failure was very low in this trial, supporting our choice of target CD34 cell dose.

The incidence of severe or life-threatening conditioning regimen—related toxicity was relatively low in this trial, suggesting that the regimen was generally well tolerated despite the median patient age of 48.5 years. The most common early toxicity was mucositis. The most serious toxicity was pulmonary-related, typically occurring between 30-100 days post-HCT. Within the first 100 days after HCT, 14% of the patients experienced grade 3-5 pulmonary toxicity,

possibly attributed to the conditioning regimen. Idiopathic pneumonia syndrome is a well-described regimen-related toxicity associated with various highdose conditioning regimens [29]. Overall, the rate of pulmonary complications observed in this trial was similar to or lower than the rates reported in previous trials in which myeloablative conditioning was used [30,31]. Interestingly, 1 study found an association between TCD and a lower risk of pulmonary complications compared with standard GVHD prophylaxis [31].

The rates of bacterial, fungal, and CMV infections encountered in the present trial did not appear to be any greater than those reported after conventional PBSC transplantation [1,32]. The low rates of acute and chronic GVHD and the absence of immunosuppressive drugs also may play roles in limiting opportunistic infections [33]. One exception is the risk of EBV reactivation, which is increased in recipients of T cell-depleted

0 7 14 21 28 35 42 49 56 63 70 77 84 91

Days Post Transplant

co 700) T3

£ 60 -a

15 50 0)

£ 40 i

0 7 14 21 28 35 42 49 56 63 70 77 84 91 Days Post Transplant

4 8 12 16

Month Post Transplant

Figure 2. Cumulative incidence of grade II-IV acute GVHD (A), grade III-IV acute GVHD (B), and extensive/limited chronic GVHD (C).

Months Post Transplant Figure 3. Cumulative incidence of relapse for all transplant recipients.

transplants [34]. Eightpatients (18%) developed evidence of EBV viremia requiring at least 1 dose of rituximab, and 1 patient died from PTLD. Thus, close monitoring for EBV infection is imperative at least up to day +100 in recipients of CD34+-enriched allografts, because early

treatment with rituximab may be able to reverse this condition in a high proportion of cases.

Relapse remains the major cause of death in patients receiving allografts for high-risk AML [1,2,4,5,35]. Because CD34 enrichment results in a TCD of 4-5 logs, there is a concern that it might decrease the putative graft-versus-leukemia effect [36]. Although this has been demonstrated clearly in patients with chronic myelogenous leukemia, there is no convincing evidence of higher relapse rates after TCD HCT for AML in remission [10,36-38]. The cumulative incidence of relapse in CR1 of <20% at 3 years suggests either the retention of a graft-versus-leukemia effect or, alternatively, the highly effective antileukemic nature of the conditioning regimen used. It is more difficult to comment on the observed risk of relapse in patients with AML in CR2, because of our low patient numbers. Only 7 patients in this trial underwent HCT while in CR2, 4 of whom relapsed. These results are in contrast to previous reports suggesting favorable outcomes after TCD HCT for AML patients in CR2 [13,14,39]. We cannot draw any definitive conclusions for this group at this time; further investigation is needed to define the relapse risk for this population.

100 80 60 40 20 0

100 80 60 40 20 0

+H—HH-

All 44 Patients 95% CI for Survival

4 8 12 16 20 24 28 32 36 Months Post Transplant

-All 44 Patients

---95% CI for Survival

~~i-1-1-1-1-1-1-1-r

4 8 12 16 20 24 28 32 36 Months Post Transplant

100 ■

r 80 ■

£ u W

Vi 1 -Mir

i i i i ~i i —n+

- -+- -------1- CR1 (N=37)

--CR2 (N=7)

4 8 12 16 20 24 28 32 36 Months Post Transplant

100 ■ 80 60 40 20

v 1 1 4-m—n+ -------1-

-CR1 (N=37)

--CR2 (N=7)

4 8 12 16 20 24 28 32 36 Months Post Transplant

Figure 4. Kaplan-Meier estimates of DFS for all transplant recipients (A), DFS by remission state (B), OS for all transplant recipients (C), and OS by remission state (D).

Although the transplantation of PBSCs may mitigate complications early after HCT, late complications related to chronic GVHD appear to be more common. A recent meta-analysis of 9 randomized trials comparing PBSCs and bone marrow clearly demonstrated a higher risk of extensive chronic GVHD after transplantation of HLA-matched sibling mobilized PBSCs [8]. The chronic GVHD observed after PBSC HCT may be more difficult to treat and may require a greater duration of corticosteroid use compared with that seen in bone marrow HCT, resulting in a higher risk of late complications [6,40]. In this context, the very low risk of extensive chronic GVHD observed in the present trial suggests another potential advantage of this approach. The low risk of both relapse and chronic GVHD makes this approach attractive for selected high-risk patients with AML in remission.

In conclusion, this study demonstrates that TCD HCT after intensive myeloablative chemoradiother-apy conditioning can be performed successfully in a multicenter setting using a single TCD method without additional posttransplantation pharmacologic GVHD prophylaxis. The low incidences of relapse and of acute and chronic GVHD in the absence of posttransplantation prophylaxis are especially encouraging. A second study to confirm these findings in unrelated donor HCT is currently being planned by the Blood and Marrow Transplant Clinical Trials Network.

ACKNOWLEDGMENTS

This work was supported in part by the National Heart, Lung, and Blood Institute and the National Cancer Institute, along with contributions from Milte-nyi Biotec.

Authorship statement: StevenM. Devine designed and performed research, analyzed and interpreted data, and wrote the manuscript. Shelly Carter designed the research, performed statistical analysis, analyzed and interpreted the data, and wrote the manuscript. Robert J. Soiffer designed and performed research, analyzed and interpreted data, and wrote the manuscript. Marcelo C. Pasquini performed research, analyzed and interpreted data, and wrote the manuscript. Parameswaran N. Hari performed research and wrote the manuscript. Anthony Stein performed research. Hillard M. Lazarus performed research and wrote the manuscript. Charles Linker performed research. Edward A. Stadtmauer performed research. Edwin P. Alyea III performed research. Carolyn A. Keever-Taylor designed and performed research, analyzed and interpreted data, and wrote the manuscript. Richard J. O'Reilly designed and performed research, analyzed and interpreted data, and wrote the manuscript.

Financial disclosure: Steven M. Devine, Shelly Carter, Robert J. Soiffer, and Richard J. O'Reilly have served as advisors to Miltenyi Biotec.

REFERENCES

1. Copelan EA. Hematopoietic stem-cell transplantation. NEnglJ Med. 200б;354:1813-182б.

2. Cornelissen JJ, van Putten WLJ, Verdonck LF, et al. Results of a HOVON/SAKK donor versus no-donor analysis of myeloa-blative HLA-identical sibling stem cell transplantation in first remission acute myeloid leukemia in young and middle-aged adults: benefits for whom? Blood. 2007;109:3б58-3ббб.

3. Koreth J, Schlenk R, Kopecky KJ, et al. Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: systematic review and meta-analysis of prospective clinical trials. JAMA. 2009;301:2349-23б1.

4. SlovakML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/ Eastern Cooperative Oncology Group study. Blood. 2000;9б: 4075-4083.

5. Suciu S, Mandelli F, de Witte T, et al. Allogeneic compared with autologous stem cell transplantation in the treatment of patients younger than 4б years with acute myeloid leukemia (AML) in first complete remission (CR1): an intention-to-treat analysis of the EORTC/GIMEMAAML-10 trial. Blood. 2003;102:1232-1240.

6. Lee SJ, Vogelsang G, Flowers MED. Chronic graft-versus-host disease. Biol Blood Marrow Transplant. 2003;9:215-233.

7. Messerer D, Engel J, Hasford J, et al. Impact of different postremission strategies on quality of life in patients with acute my-eloid leukemia. Haematologica. 2008;93:82б-833.

8. Stem Cell Trialists' Collaborative Group. Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol. 2005;23:5074-5087.

9. Champlin RE, Passweg JR, Zhang M-J, et al. T-cell depletion of bone marrow transplants for leukemia from donors other than HLA-identical siblings: advantage of T-cell antibodies with narrow specificities. Blood. 2000;95:399б-4003.

10. Ho VT, Soiffer RJ. The history and future of T-cell depletion as graft-versus-host disease prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood. 2001;98:3192-3204.

11. Aversa F, Terenzi A, Tabilio A, et al. Full haplotype-mismatched hematopoietic stem-cell transplantation: a phase II study in patients with acute leukemia at high risk of relapse. J Clin Oncol. 2005;23:3447-3454.

12. Handgretinger R, Klingebiel T, Lang P, et al. Megadose transplantation of purified peripheral blood CD34(+) progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant. 2001;27:777-783.

13. Jakubowski AA, Small TN, Young JW, et al. T cell-depleted stem cell transplantation for adults with hematologic malignancies: sustained engraftment of HLA-matched related donor grafts without the use of antithymocyte globulin. Blood. 2007; 110:4552-4559.

14. Papadopoulos EB, Carabasi MH, Castro-Malaspina H, et al. T-cell-depleted allogeneic bone marrow transplantation as postremission therapy for acute myelogenous leukemia: freedom from relapse in the absence of graft-versus-host disease. Blood. 1998;91:1083-1090.

15. Soiffer RJ, Fairclough D, Robertson M, et al. CD6-depleted al-logeneic bone marrow transplantation for acute leukemia in first complete remission. Blood. 1997;89:3039-3047.

16. Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol. 2003;21:4б42-4б49.

17. Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood. 2002;100:2292-2302.

18. Niesters HGM, van Esser J, Fries E, et al. Development of a real-time quantitative assay for detection of Epstein-Barr virus. J Clin Microbiol. 2000;38:712-715.

19. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease, I: Diagnosis and Staging Working Group report. Biol Blood Marrow Transplant. 2005; 11:945-956.

20. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995;15:825-828.

21. ByrdJC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002;100:4325-4336.

22. Baldus CD, Thiede C, Soucek S, et al. BAALC expression and FLT3 internal tandem duplication mutations in acute myeloid leukemia patients with normal cytogenetics: prognostic implications. J Clin Oncol. 2006;24:790-797.

23. Gale RE, Green C, Allen C, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood. 2008;111: 2776-2784.

24. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958;53:457-481.

25. Gooley TA, Leisenring W, Crowley J, et al. Estimation of failure probabilities in the presence of competing risks: new representations of old estimators. Stat Med. 1999;18:695-706.

26. Bornhauser M, Platzbecker U, Theuser C, et al. CD34+-en-riched peripheral blood progenitor cells from unrelated donors for allografting of adult patients: high risk of graft failure, infection and relapse despite donor lymphocyte add-back. Br J Haematol. 2002;118:1095-1103.

27. Elmaagacli AH, Peceny R, Steckel N, et al. Outcome of transplantation of highly purified peripheral blood CD341 cells with T-cell add-back compared with unmanipulated bone marrow or peripheral blood stem cells from HLA-identical sibling donors in patients with first chronic phase chronic myeloid leukemia. Blood. 2003;101:446-453.

28. Kernan NA, Collins NH, Juliano L, et al. Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-v-host disease. Blood. 1986;68:770-773.

29. Cooke KR, Yanik G. Acute lung injury after allogeneic stem cell transplantation: is the lung a target of acute graft-versus-host disease? Bone Marrow Transplant. 2004;34:753-765.

30. Fukuda T, Hackman RC, Guthrie KA, et al. Risks and outcomes of idiopathic pneumonia syndrome after nonmyeloablative and conventional conditioning regimens for allogeneic hematopoi-etic stem cell transplantation. Blood. 2003;102:2777-2785.

31. Ho VT, Weller E, Lee SJ, et al. Prognostic factors for early severe pulmonary complications after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2001;7: 223-229.

32. Junghanss C, Marr KA, Carter RA, et al. Incidence and outcome of bacterial and fungal infections following nonmyeloablative compared with myeloablative allogeneic hematopoietic stem cell transplantation: a matched control study. Biol Blood Marrow Transplant. 2002;8:512-520.

33. van Burik J-AH, Carter SL, Freifeld AG, et al. Higher risk of cytomegalovirus and aspergillus infections in recipients of T cell—depleted unrelated bone marrow: analysis of infectious complications in patients treated with T cell depletion versus immunosuppressive therapy to prevent graft-versus-host disease. Biol Blood Marrow Transplant. 2007;13:1487-1498.

34. Landgren O, Gilbert ES, Rizzo JD, et al. Risk factors for lym-phoproliferative disorders after allogeneic hematopoietic cell transplantation. Blood. 2009;113:4992-5001.

35. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med. 2008;358:1909-1918.

36. Marmont AM, Horowitz MM, Gale RP, et al. T-cell depletion of HLA-identical transplants in leukemia. Blood. 1991;78:2120-2130.

37. Goldman JM, Gale RP, Horowitz MM, et al. Bone marrow transplantation for chronic myelogenous leukemia in chronic phase. Increased risk for relapse associated with T-cell depletion. Ann Intern Med. 1988;108:806-814.

38. Wagner JE, Thompson JS, Carter SL, et al. 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. Lancet. 2005;366:733-741.

39. Aversa F, Terenzi A, Carotti A, et al. Improved outcome with T-cell—depleted bone marrow transplantation for acute leukemia. J Clin Oncol. 1999;17:1545-1550.

40. Flowers MED, Parker PM, Johnston LJ, et al. Comparison of chronic graft-versus-host disease after transplantation of peripheral blood stem cells versus bone marrow in allogeneic recipients: long-term follow-up of a randomized trial. Blood. 2002; 100:415-419.