Prospective Multicenter Study of Single-Unit Cord Blood Transplantation with Myeloablative Conditioning for Adult Patients with High-Risk Hematologic Malignancies Academic research paper on "Clinical medicine"

Prospective Multicenter Study of Single-Unit Cord Blood Transplantation with Myeloablative Conditioning for Adult Patients with High-Risk Hematologic Malignancies

Takehiko Mori1,*, Masatsugu Tanaka2, Takeshi Kobayashi3, Kazuteru Ohashi3, Shin Fujisawa4, Akira Yokota5, Hiroyuki Fujita6, Chiaki Nakaseko 7, Toru Sakura 8, Yasuhito Nannya 9, Satoshi Takahashi10, Heiwa Kanamori2, Yoshinobu Kanda11, Hisashi Sakamaki3, Shinichiro Okamoto1, for the Kanto Study Group for Cell Therapy

1 Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan

2 Department of Hematology, Kanagawa Cancer Center, Kanagawa, Japan

3 Department of Hematology, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan

4 Department of Hematology, Yokohama City University Medical Center, Yokohama, Japan

5 Division of Hematology, Department of Internal Medicine, Chiba Aoba Municipal Hospital, Chiba, Japan

6 Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan

7 Department of Hematology, Chiba University Graduate School of Medicine, Chiba, Japan

8 Division of Hematology, Saiseikai Maebashi Hospital, Gunma, Japan

9 Department of Hematology and Oncology, University of Tokyo, Tokyo, Japan

10 Department of Hematology and Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

11 Division of Hematology, Saitama Medical Center, Jichi Medical University, Saitama, Japan

American Society for Blood and Marrow Transplantation

Article history:

Received 22 August 2012

Accepted 12 December 2012

Key Words: Total body irradiation Graft-versus-host disease Non-relapse mortality Acute leukemia

ABSTRACT

Although the use of cord blood transplantation (CBT) is increasing, the optimal methods for conditioning and graft-versus-host disease (GVHD) prophylaxis remain to be established. Among previous reports, the Institute of Medical Science, University of Tokyo (IMSUT) has reported remarkably favorable results of CBT for hematologic malignancies as a single-institute experience. The aim of the present multicenter prospective study was to assess the safety and efficacy of CBT performed precisely according to IMSUT transplantation procedures. Thirty-three adult patients with hematologic malignancies, such as acute leukemia, chronic myelogenous leukemia, or myelodysplastic syndrome, either lacking an HLA-identical sibling/HLA-matched unrelated donor or requiring urgent transplantation were enrolled. Conditioning consisted of total body irradiation (12 Gy), cytarabine, and cyclophosphamide. Cyclosporine A and methotrexate were used for GVHD prophylaxis. Diagnoses were acute leukemia in 26 patients, chronic myelogenous leukemia in 4, and mye-lodysplastic syndrome in 3; 12 patients were in first complete remission, and the others were in advanced stages at the time of CBT. Thirty-one patients achieved engraftment, and the cumulative incidence of grade II-IV acute GVHD was 45% (95% confidence interval, 28%-62%). With a median follow-up of 46.2 months in 16 surviving patients, the 1-year cumulative incidence of nonrelapse mortality was 15% (95% confidence interval, 5%-30%). Causes of nonrelapse mortality were infection (n = 4) and graft failure (n = 1). The overall and disease-free survival rates were 51% (95% CI, 34%-68%) and 42% (95% CI, 26%-59%), respectively. These results suggest that the IMSUT CBT procedures can safely provide a high disease-free survival rate in patients with high-risk hematologic malignancies.

© 2013 American Society for Blood and Marrow Transplantation.

INTRODUCTION

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most promising curative treatment for hema-tologic malignancies. Several hematopoietic stem cell sources are now available, and the use of cord blood transplantation (CBT) has been increasing dramatically [1]. However, the outcomes of CBT are not necessarily satisfactory, because of the high nonrelapse mortality (NRM). Conditioning and prophylaxis against graft-versus-host disease (GVHD) used in CBT have varied significantly among previous studies, and the optimal approaches remain to be established [2-6]. Among those studies, the outcomes

Financial disclosure: See Acknowledgments on page 490.

* Correspondence and reprint requests: Takehiko Mori, MD, PhD, Division of Hematology, Department of Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: tmori@a3.keio.jp (T. Mori).

1083-8791/$ — see front matter © 2013 American Society for Blood and Marrow http://dx.doi.org/10.1016/j.bbmt.2012.12.007

of CBT for hematologic diseases at the Institute of Medical Science, University of Tokyo (IMSUT) were notably favorable and in fact were superior to the outcomes of allogeneic bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT) from related and unrelated donors at the same institution [7,8].

The IMSUT transplantation procedures involve a myeloa-blative conditioning regimen using total body irradiation (TBI), cyclophosphamide (CY), and high-dose cytarabine. Cytarabine is combined with granulocyte colony-stimulating factor (G-CSF) for myeloid malignancies. In addition, cyclo-sporine A is given over 10 hours with short-term metho-trexate (MTX) for GVHD prophylaxis. To date, however, no study has systematically assessed whether IMSUT's favorable results would be reproduced if CBT for hematologic malignancy were performed precisely according to IMSUT's transplantation procedures. Accordingly, we designed and performed a multi-institutional study to evaluate the safety Transplantation.

and efficacy of single-unit CBT for hematologic malignancies by strictly following these procedures.

PATIENTS AND METHODS

This study is a multi-institutional prospective study of the Kanto Study Group for Cell Therapy. The protocol was approved by the Institutional Review Boards of the 9 participating institutions and registered at http:// clinicaltrials.gov (NCT00270881). No patients were enrolled from IMSUT. Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki.

Patient Eligibility and Cord Blood Unit Selection

Eligibility criteria for this study included (1) age 20-55 years; (2) diagnosis of acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), or myelodysplastic syndrome (mDS) suitable for allogeneic HSCT; (3) lack of an available 5/6 or fully HLA-matched related donor; (4) either lack of an available fully 6/6 HLA-matched unrelated donor or the need for immediate HSCT based on the features of the disease as judged by the treating physician; (5) availability of a 6/6, 5/6, or 4/6 serologically HLA-matched cord blood unit with a minimum of 2 x 107 total nucleated cells per kilogram of recipient body weight before cryopreservation in the Japan Cord Blood Bank Network; (6) Eastern Cooperative Oncology Group performance status of 0 or 1; (7) adequate function of main organs, including the liver, kidneys, lungs, and heart; and (8) lack of anti-HLA (class I and/or II) antibody. Patients with a previous history of HSCT, active infection, or active central nervous system disease or psychiatric disorders were excluded. This protocol was only for patients receiving single-unit CBT. HLA disparity was determined based on the antigen level of HLA-A, -B, and -DR loci specified by low- or high-resolution techniques.

Conditioning Regimen

All patients received the same myeloablative conditioning as described previously [7-9]. TBI 12 Gy was delivered in 4 or 6 fractions for 2 or 3 days (days -8, -7, and -6 or days -7 and -6). After completion of TBI, cytarabine at a dose of 2 or 3 g/m2 was administered i.v. over 2 hours every 12 hours for 2 consecutive days (days -5 and -4). All patients received steroid eye drops for prophylaxis against keratoconjunctivitis due to cytarabine. For myeloid malignancies (AML, MDS, and CML), recombinant human granulocyte colony-stimulating factor (G-CSF; lenograstim) was given by continuous infusion at a daily dose of 5 mg/kg, starting 12 hours before the first dose of cytarabine and continuing until the last dose of cytarabine. Then CY 60 mg/ kg was administered i.v. over 2 hours for 2 consecutive days (days -3 and -2). Cytarabine could be omitted in exceptional cases based on factors in the patient's background, such as a history of allergic reaction. No patient received antithymocyte globulin as part of the conditioning regimen.

Infusion of Cord Blood, GVHD Prophylaxis, and Supportive Care

Two days after the completion of CY administration (day 0), patients received CBT. The cord blood graft was thawed and immediately infused without washing. GVHD prophylaxis was provided with short-term methotrexate (MTX; 15 mg/m2 on day 1, and 10 mg/m2 on days 3 and 6) and cyclosporine A (CsA). Leucovorin was given i.v. to ameliorate its toxicity. CsA was given i.v. over 10 hours starting on day -1. The CsA dose was adjusted at the discretion of the physician only if the trough level of CsA was <100 ng/mL or adverse events associated with CsA developed.

Each patient was isolated in a laminar air-flow or high-efficiency particulate air-filtered room. The administration of lenograstim at a dose of 5 mg/kg was started 1 day after CBT and continued until neutrophil recovery was achieved. Prophylactic fluoroquinolone and fluconazole (200 mg/day) were given orally, starting 14 days before transplantation. For Pneumocystis pneumonia prophylaxis, cotrimoxazole was given for 14 consecutive days before transplantation and recommenced on a schedule of 2-3 days per week after sustained hematopoietic recovery was confirmed. Oral acyclovir at a dose of 1000 mg/day was given from day -7 to day 35. Cytomegalovirus (CMV) reactivation was routinely monitored by CMV antigenemia assay or PCR soon after neutrophil recovery, which triggered preemptive therapy with ganciclovir. Intravenous immunoglobulin was given in patients with a serum immunoglobulin G level <500 mg/dL.

Assessment of Chimerism, Engraftment, and GVHD

The chimerism study was performed on whole bone marrow cells at 1 month, 2 months, and 3 months after CBT. Analyses were performed by fluorescein in situ hybridization for X and Y chromosomes or by microsatellite PCR as appropriate. The day of myeloid engraftment was defined as the first day of 3 consecutive days when the absolute neutrophil count exceeded 0.5 x 109/L. The day of platelet engraftment was defined as the day

Table 1

Patient and Transplant Characteristics (n = 33)

Characteristic Value

Age, years, median (range) 37 (21-54)

Sex, males/females, n 21/12

Body weight, kg, median (range) 55.0 (39.1-97.0) Diagnosis, n

AML 20

MDS 3 Disease status, n

CR1 12

CR2, chronic phase 2 5

Not in CR, blast crisis 16 Conditioning, n

TBI + cytarabine + G-CSF + CY 27

TBI + cytarabine + CY 5

TBI + CY 1 Cord blood units HLA disparity, n

4-antigen match 29

5-antigen match 3

6-antigen match 1 Nucleated cells per kg body weight, median (range) 2.66 (2.00-4.58)

GVHD prophylaxis with CsA + short-term MTX, n 33

when the absolute platelet count exceeded 20 x 109/L without platelet transfusion. Primary graft failure was defined as lack of myeloid engraftment until day 42; secondary graft failure, as a persistent loss of myeloid engraftment after having achieved engraftment. Both acute and chronic GVHD were diagnosed and graded based on published criteria [10,11].

Statistical Analysis

The primary endpoint of this study was 1-year NRM, and secondary endpoints were engraftment, acute and chronic GVHD, infectious complications, day +100 NRM, relapse rate, disease-free survival (DFS), and overall survival (OS). Survival rates were calculated by the Kaplan-Meier method. Probability of acute GVHD, disease relapse, and NRM were estimated on the basis of cumulative incidence curves to accommodate the following competing events: death without GVHD and second transplantation for graft failure for acute GVHD, death for relapse, and relapse for NRM [12]. Comparisons were made using the log-rank test or Gray test as appropriate. Multivariate analyses were performed using the Cox proportional hazards model or the Fine and Gray proportional-hazards model as appropriate. P < .05 was considered to indicate statistical significance in all analyses.

RESULTS

Patient Characteristics

Thirty-three patients were enrolled and underwent CBT. Patient and transplant characteristics are summarized in Table 1. At the time of CBT, 12 patients with AML/ALL were in the first complete remission (CR1) and were defined as standard-risk patients. The remaining 19 patients had AML/ALL in CR2 or CML in chronic phase 2 (n = 3) or AML/ALL not in remission, MDS with an excess of blasts, or CML in blastic crisis (n = 16), and were defined as high-risk patients. Three patients in CR1 had Philadelphia chromosome—positive ALL. All but 1 patient received TBI, cytarabine, and CY with or without G-CSF as conditioning.

Engraftment and Chimerism

Myeloid engraftment was obtained at a median of 26 days (range, 18-60 days) in 31 patients. Platelet engraftment was obtained at a median of 44 days (range, 25-140 days) in 26 patients. Two patients experienced primary graft failure, caused by graft rejection in 1 patient and early disease progression in 1 patient. One case of secondary graft failure occurred after hemophagocytic syndrome. In the 27 patients who underwent chimerism analysis, full donor chimerism

Table 2

Infectious Complications

Infection Number

Bacterial (n = 14)

Bacteremia 10

Enteritis 1

Meningitis 1

Pneumonia 1

Cholecystitis 1

Fungal (n = 5)

Candidemia 1

Invasive aspergillosis 3

Pneumocystis pneumonia 1

Viral (n = 28)

CMV infection 23

CMV disease 1

HHV-6 central nervous system disorder 1

Parainfluenza virus pneumonia 1

Encephalitis* 2

HHV-6 indicates human herpesvirus 6.

* Causative virus was not identified.

was obtained at 1 month posttransplantation in 24 patients, at 2 months in 2 patients, and at 3 months in 1 patient.

Acute and Chronic GVHD

Acute GVHD developed in 21 of 31 evaluable patients with myeloid engraftment (grade I in 6 patients, grade II in 10, grade III in 3, and grade IV in 2). The cumulative incidence of grade II-IV acute GVHD up to day +100 posttransplantation was 46% (95% CI, 27.8%-61.5%). Among the 10 patients with grade II acute GVHD, 6 did not require systemic glucocorticoid in addition to CSA for the treatment of acute GVHD. Among the 27 patients who survived more than 100 days after transplantation, 6 patients developed chronic GVHD (2 with extensive type and 4 with limited type).

Infectious Complications

All but 1 patient experienced at least 1 episode of infectious complications after CBT (Table 2). The most common infective pathogen was viruses, including CMV infection, followed by bacteria and fungus. Four cases of infectious complications were fatal (2 with bacteremia and 2 with encephalitis). All of these patients had grade II-IV acute GVHD (1 with grade II, 1 with grade III, and 2 with grade IV) and were receiving systemic glucocorticoid therapy when infectious complications developed.

NRM, Relapse, and Survival

At a median follow-up of 46.2 months (range, 31.0-65.8 months), 16 patients were alive. Causes of death in the other 17 patients included relapse and complications associated with treatment of relapse after transplantation (12 patients), infectious complications (4 patients), and graft failure (1 patient). The cumulative incidence of NRM was 9% (95% CI, 2%-22%) at 100 days post-HSCTand 15% (95% CI, 5%-30%) at 1 year post-HSCT (Figure 1A). The cumulative incidence of NRM at 3 years post-HSCT did not differ significantly between standard-risk and high-risk patients (25% versus 10%; P = .254). In 14 patients, disease relapse or progression occurred at a median of 9 months (range, 0.9-23.0 months) post-HSCT. The 3-year relapse rate was 42% (95% CI, 25%-59%) (Figure 1B). OS was 51% (95% CI, 34%-68%), and DFS was 42% (95% CI, 26%-59%) (Figure 1C). The 16 patients who were alive without disease remained in good condition, with an Eastern Cooperative Oncology Group performance status

.c a .c

-I—I-

I II 11

—I— l2

—I—

—I—

—I—

—I— 60

Months after transplantation

24 36 48 60 72 Months after transplantation

Overall survival

Disease-free survival

12 24 36 48 60 Months after transplantation

Figure 1. Cumulative incidences of NRM (A) and relapse rate (B), and KaplanMeier estimates of OS and DFS (C). "+" indicates a censored patient.

of 0 (n = 13) or 1 (n = 3). Two of the 3 patients who experienced primary or secondary graft failure and 4 of 14 patients who experienced disease relapse or progression subsequently underwent a second allogeneic HSCT. At the

time of this analysis, 2 patients were still alive without disease at 23 months and 62 months after the second HSCT.

As the possible factors affecting the rates of disease relapse, NRM, OS, and DFS, the following variables were analyzed: patient age (<40 years versus >40 years) and sex, risk categories based on disease status at transplantation (standard risk versus high risk), HLA disparity (4/6 versus 5/6 and full match), nucleated cell dose of cord blood graft (<2.60 x 107/kg versus >2.60 x 107/kg), and development of acute GVHD (none or grade I versus grade II-IV). The development of grade II-IV acute GVHD had a negative impact on NRM (1-year NRM, 27% [95% CI, 8%-51%] versus 0%; P < .05); however, none of other variables, including patient age, had a significant impact on NRM, disease relapse, OS, and DFS.

DISCUSSION

The results of this prospective multicenter study demonstrate that single-unit CBT following myeloablative conditioning can provide favorable survival with low NRM in adult patients with high-risk hematologic malignancies. Our study is unique in that all patients received uniform conditioning, GVHD prophylaxis, and other supportive care. All of the CBT procedures applied in this study were identical to those reported by the IMSUT. In the IMSUT reports, the outcome of CBT was superior to that of allogeneic BMT or PBSCT from related and unrelated donors, which provided a notably favorable outcome compared with other studies [7,8]. Given the nature of the retrospective single-center study, we deemed it necessary to confirm the reproduc-ibility of the results, and consequently planned and conducted a prospective multicenter study to evaluate the safety and efficacy of the IMSUT transplantation procedures. Of note, our study enrolled patients with high-risk hematologic malignancies regardless of disease status at transplantation, and indeed half of the patients enrolled were not in remission at the time of transplantation. Despite our use of such a high-risk cohort, the primary endpoint of 1-year NRM was 15%, which was comparable with the 9% reported by IMSUT and lower than those of other studies (30%~60%) [4-6,13-15]. The primary cause of NRM in the present study was infectious complications occurring after engraftment. All of those infectious complications occurred in patients who developed grade II-IV acute GVHD, and the sole factor significantly associated with the incidence of NRM was grade II-IV acute GVHD. Thus, to further reduce NRM, the optimal management of infectious complications should be explored, particularly in patients developing acute GVHD after CBT.

Disease relapse greatly interferes with the success of allogeneic HSCT, especially in patients with chemorefractory hematologic malignancies, and the intensity of conditioning plays a crucial role. Although TBI plus CY (TBI-CY) remains the most common myeloablative conditioning regimen for allogeneic HSCT, further intensification of conditioning by administering additional antileukemic agents has been attempted in an effort to reduce disease relapse. Although this further intensification can lead to more effective disease control, the benefit is generally offset by the higher rates of NRM, and thus the effect of this approach on survival remains controversial [16]. Cytarabine has been extensively investigated as an additional agent in this setting, but this drug is also associated with significant toxicity [17-22]. However, in a recent study we found favorable outcomes with low NRM with the use of TBI-CY plus cytarabine as conditioning in patients with ALL [9]. Based on these findings and the fact that the IMSUT protocol uses mainly TBI-CY plus

cytarabine as conditioning for CBT [7,8], we followed the same regimen. In addition, we combined cytarabine with G-CSF infusion in patients with myeloid malignancies, based on the hypothesis that G-CSF increases the susceptibility of myeloid leukemic cells to cytarabine, thereby contributing to decreased relapse rate [23-30]. In the setting of allogeneic BMT and PBSCT, favorable outcomes of the conditioning consisting of TBI and G-CSF with cytarabine in patients with AML and advanced MDS have been reported [31-34]. In the present study, despite the high-risk features of the disease, approximately half of the patients achieved long-term DFS with this unique intensified myeloablative conditioning because of the low NRM and disease relapse, as expected, which seemed more favorable than the results of previous studies [5,14]. These results suggest that the myeloablative conditioning regimen used in the present study is safe and highly effective in eradicating leukemic cells even in patients with high-risk leukemia. However, the outcomes of our study patients seem inferior to those reported by IMSUT in terms of disease relapse and survival. The major difference is in the incidence of disease relapse, which was higher in the present study (40% versus 16%~17%) and consequently had an effect on DFS (42% versus 70%~74%). The most plausible explanation for this difference is the differing study designs (prospective multicenter versus retrospective single-center). Another possible explanation may be related to demographic differences in the patient cohorts. The standard variables, such as age and disease status at transplantation, seemed similar in the 2 studies. However, BMT from unrelated donors takes priority over CBT in clinical practice in all of the institutions participating in the present study. Thus, it is possible that high-risk patients, particularly those with diseases in CR, might be selectively enrolled into the present study based on the inclusion criteria defining the requirement of immediate HSCT based on disease features at the discretion of each treating physician. Disease status at transplantation cannot precisely reflect the risk of such features of the disease and could have significantly affected the outcome.

Along with conditioning, GVHD prophylaxis plays an important role in the outcomes of allogeneic HSCT. However, a standard GVHD prophylaxis regimen for CBT has yet to be established, and GVHD prophylaxis varies widely among previous studies, variously consisting of CsA or tacrolimus alone or in combination with MTX, glucocorticoids, myco-fenolate mofetil (MMF), and/or antithymocyte globulin [2-6,13-15,35]. To avoid the toxicity and negative effects of MTX on hematopoietic reconstitution, several studies have applied non-MTX-containing GVHD prophylaxis. In contrast, CsA in combination with short-term MTX, which remains a common GVHD prophylaxis regimen in allogeneic HSCTs other than CBT, was used in the present study precisely according to the IMSUT protocol. Leucovorin was routinely given to ameliorate the toxicity of MTX. In addition, CsA was given over 10 hours at a dose of 3 mg/kg/day, with the dose adjusted only when the CsA trough level was <100 ng/mL or adverse events associated with CsA developed. With this relatively unique regimen, neutrophil engraftment was obtained at a median of 26 days after CBT, which is comparable with those in previous studies. The incidence of grade II-IV acute GVHD of 48% is identical to that reported by IMSUT (44%~52%) [7,8]. Of note, however, acute GVHD was manageable without systemic glucocorticoid administration in approximately 60% of patients with grade II acute GVHD, suggesting that the incidence of clinically

significant acute GVHD requiring systemic glucocorticoid therapy was actually lower with this prophylactic regimen. In addition, the development of acute GVHD requiring systemic glucocorticoid therapy was associated with NRM due to fatal infectious complications. Therefore, further investigations should focus on the more effective GVHD prophylaxis, although the balance between the immuno-suppressive effect and graft-versus-tumor effect is also important. In this setting, less-toxic GVHD prophylaxis using MMF instead of MTX could be a good option, because it has a potential to allow faster engraftment and could decrease the risk of infectious complications.

Secondary graft failure due to graft rejection was observed in 1 patient, who was rescued by the second CBT and alive at 26 months after the second CBT. Other complications interfering with survival and quality of life, such as severe chronic GVHD and secondary malignancies, were seen in no patients during the follow-up period. All surviving patients were in good clinical condition without disease recurrence. These results demonstrate the long-term safety and tolerability of these CBT procedures despite the highly intense conditioning.

Two major limitations of the present study are the small number of patients evaluated and the heterogeneous disease background in these patients, including the types of disease, high-risk features, and disease status at transplantation. However, the study's primary endpoint was to evaluate 1-year NRM with respect to safety of the transplantation procedures. Thus, we believe that safety of the procedures can be confirmed even with our relatively small, heterogeneous cohort.

We conclude that the transplantation methods used in the present study, including intensified myeloablative conditioning, CsA with MTX as GVHD prophylaxis, and other supportive care measures, can provide low NRM and high survival rates in patients undergoing single-unit CBT for hematologic malignancies and could possibly become a standard treatment. However, because of the limited number of patients and high incidence of life-threatening infectious complications associated with GVHD, a larger-scale study with some modifications to GVHD prophylaxis is needed to establish the optimal CBT techniques.

ACKNOWLEDGMENTS

We are indebted to all of the staff members of the collaborating institutes and the data center of the Kanto Study Group for Cell Therapy for their assistance. We thank Dr Hisashi Gondo, Dr Shigehisa Mori, and Dr Yuji Tanaka for monitoring the safety and efficacy of this study.

Financial disclosure: There are no conflicts of interest to report.

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