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Mixed Chimerism and Secondary Graft Failure in Allogeneic Hematopoietic Stem Cell Transplantation for Aplastic Anemia
Secondary graft failure in transplant for aplastic anemia
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Mixed chimerism and secondary graft failure in allogeneic hematopoietic stem cell transplantation for aplastic anemia Shinichi Kako M.D. , Hirohito Yamazaki M.D. , Kazuteru Ohashi M.D. , Yukiyasu Ozawa M.D. , Shuichi Ota M.D. , Yoshinobu Kanda M.D. , Tetsuo Maeda M.D. , Jun Kato M.D. , Ken Ishiyama M.D. , Ken-ichi Matsuoka M.D. , Toshihiro Miyamoto M.D. , Hiroatsu Iida M.D. , Kazuhiro Ikegame M.D. , Takahiro Fukuda M.D. , Tatsuo Ichinohe M.D. , Yoshiko Atsuta M.D. , Takehiko Mori M.D. , on behalf of the Adult Aplastic Anemia Working Group of the Japanese Society for Hematopoietic Cell Transplantation PII: DOI: Reference:
S1083-8791(19)30660-3 https://doi.org/10.1016/j.bbmt.2019.10.004 YBBMT 55752
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
6 August 2019 1 October 2019
Please cite this article as: Shinichi Kako M.D. , Hirohito Yamazaki M.D. , Kazuteru Ohashi M.D. , Yukiyasu Ozawa M.D. , Shuichi Ota M.D. , Yoshinobu Kanda M.D. , Tetsuo Maeda M.D. , Jun Kato M.D. , Ken Ishiyama M.D. , Ken-ichi Matsuoka M.D. , Toshihiro Miyamoto M.D. , Hiroatsu Iida M.D. , Kazuhiro Ikegame M.D. , Takahiro Fukuda M.D. , Tatsuo Ichinohe M.D. , Yoshiko Atsuta M.D. , Takehiko Mori M.D. , on behalf of the Adult Aplastic Anemia Working Group of the Japanese Society for Hematopoietic Cell Transplantation, Mixed chimerism and secondary graft failure in allogeneic hematopoietic stem cell transplantation for aplastic anemia, Biology of Blood and Marrow Transplantation (2019), doi: https://doi.org/10.1016/j.bbmt.2019.10.004
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Society for Transplantation and Cellular Therapy
Highlights
The influence of MC and/or SGF in HSCT for AA was retrospectively evaluated.
SGF with both MC/recipient- and donor-type chimerism was observed.
Patients who developed SGF with both types of chimerism had unfavorable outcomes.
The use of fludarabine may affect the occurrence of SGF with both types of chimerism.
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Regular Manuscript
Mixed chimerism and secondary graft failure in allogeneic hematopoietic stem cell transplantation for aplastic anemia
Shinichi Kako, M.D.1, Hirohito Yamazaki, M.D.2, Kazuteru Ohashi, M.D.3, Yukiyasu Ozawa, M.D.4, Shuichi Ota, M.D.5, Yoshinobu Kanda, M.D.1, Tetsuo Maeda, M.D.6, Jun Kato, M.D.7, Ken Ishiyama, M.D.8, Ken-ichi Matsuoka, M.D.9, Toshihiro Miyamoto, M.D.10, Hiroatsu Iida, M.D.11, Kazuhiro Ikegame, M.D.12, Takahiro Fukuda, M.D.13, Tatsuo Ichinohe, M.D.14, Yoshiko Atsuta, M.D.15, Takehiko Mori, M.D.7, on behalf of the Adult Aplastic Anemia Working Group of the Japanese Society for Hematopoietic Cell Transplantation
1Division
of Hematology, Jichi Medical University Saitama Medical Center, Saitama,
Japan, 2Division of Transfusion Medicine, Kanazawa University Hospital, Kanazawa, Japan, 3Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan, 4Department of Hematology, Japanese Red
2
Cross Nagoya First Hospital, Nagoya, Japan, 5Department of Hematology, Sapporo Hokuyu Hospital, Sapporo, Japan, 6Department of Hematology and Oncology, Osaka University Hospital, Osaka, Japan, 7Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan,
8Department
of Hematology,
Kanazawa University Hospital, Kanazawa, Japan, 9Division of Hematology/Oncology, Okayama University Hospital, Hiroshima, Japan,
10Medicine
and Biosystemic Science,
Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan,
11Division
of Cell Therapy, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
12Division
of Hematology, Department of Internal Medicine, Hyogo College of
Medicine, Hyogo, Japan 13Hematopoietic Stem Cell Transplantation Division, National Cancer Center Hospital, Tokyo, Japan
14Department
of Hematology and Oncology,
Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan,
15Japanese
Data Center for Hematopoietic Cell Transplantation,
Nagoya, Japan
Corresponding author: Shinichi Kako, M.D. Division of Hematology, Department of Internal Medicine, Jichi Medical University Saitama Medical Center
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1-847 Amanuma, Omiya-ku, Saitama-city, Saitama 330-8503, Japan TEL: +81-48-647-2111 ext. 5881 FAX: +81-48-648-5188 E-mail: [email protected]
Short title Secondary graft failure in transplant for aplastic anemia
Conflict of interest disclosures Yamazaki H reports receiving honoraria from Sanofi K.K., and Kanda Y reports receiving research funding from Sanofi K.K. and Shionogi & CO., LTD. with regard to this study.
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Abstract Mixed chimerism (MC) and/or secondary graft failure (SGF) with recipient- or donor-type chimerism is a major obstacle in allogeneic transplantation for aplastic anemia (AA). From a registry database in Japan, patients with AA aged more than 15 years who underwent a first allogeneic bone marrow or peripheral blood transplantation between 2000 and 2014 and achieved engraftment were included in this study. MC that did not require either granulocyte-colony stimulating factor (G-CSF) or transfusion support (Group 1), MC (not SGF) that required G-CSF and/or transfusion support (Group 2), SGF with MC or complete recipient-type chimerism (Group 3), and SGF with complete donor-type chimerism (Group 4) developed in 26, 16, 19, and 17 patients, respectively. The overall median follow-up period for survivors was 1727 days. The overall survival rate (OS) was 90.4% at 1 year and 83.5% at 5 years in patients without MC or SGF (n = 340), which was not different from OS in Group 1 or 2. However, inferior OS was observed in Group 3 (1 year: 52.1%, 5 years: 52.1%) and Group 4 (1 year: 82.4%, 5 years: 56.3%). In multivariate analyses, the use of fludarabine (Flu) and the absence of irradiation in conditioning were associated with the development of SGF with MC or complete recipient-type chimerism, and the use of Flu in conditioning was associated with SGF with complete donor-type chimerism. In
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conclusion, the use of Flu may affect the occurrence of SGF with both recipient- and donor-type chimerism.
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Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective and curative treatment option for patients with aplastic anemia (AA). High-dose cyclophosphamide (CY) (200 mg/kg) combined with anti-thymocyte globulin (ATG) is frequently used as a conditioning regimen, and shows excellent outcomes especially in HSCT from a human leukocyte antigen (HLA)-matched sibling[1]. However, the cardiotoxicity of high-dose CY is a major problem[2], since patients with AA often have cardiac dysfunction due to long-term anemia and iron overload associated with massive transfusion. Recently, conditioning regimens involving fludarabine (Flu) and reduced-dose CY are increasingly being used to avoid the toxicity of high-dose CY. Good clinical courses without graft rejection in HSCT from an HLA-matched sibling have been reported[3], and even in HSCT from alternative donors, a conditioning regimen that includes Flu and reduced-dose CY can overcome graft rejection with the addition of low-dose total body irradiation (TBI)[4]. However, mixed chimerism (MC) after neutrophil engraftment and/or secondary graft failure (SGF) is still observed with such conditioning regimens, especially in HSCT from an HLA-matched sibling[5]. Moreover, SGF with complete donor-type chimerism appears to be another obstacle. In Japan, this condition has mainly been reported in pediatric patients with AA [6, 7], but
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has also been observed in adult patients with AA[5]. However, the risk factors for MC and/or SGF with recipient- or donor-type chimerism and the influence of these conditions on the prognosis remain to be elucidated, especially in adult patients with AA. Therefore, we performed a retrospective analysis using the registry database of the Japanese Society for Hematopoietic Cell Transplantation (JSHCT) to clarify the current state of MC and/or SGF after HSCT in adult patients with AA.
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Patients and Methods Data source The registry database of JSCHT is called the Transplant Registry Unified Management Program (TRUMP)[8, 9]. In TRUMP, patients with AA aged more than 15 years who underwent a first allogeneic bone marrow or peripheral blood transplantation between 2000 and 2014 and achieved neutrophil engraftment were initially selected. Next, an additional survey was conducted for these patients to collect detailed data regarding MC and SGF, and patients for whom this information was available were included in this study. This study was planned by the Adult Aplastic Anemia Working Group of JSHCT and approved by the data management committee of TRUMP and the institutional review board of Jichi Medical University Saitama Medical Center.
Definition of mixed chimerism and secondary graft failure Chimerism analyses were performed using peripheral blood or bone marrow cells. The timing and source of chimerism analyses depended on each physician’s discretion. MC was defined as the detection of at least one recipient-type cell in sex-chromosome analysis, or a rate of recipient-type cells of at least 5% in
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sex-chromosome fluorescence in situ hybridization (FISH) analysis or short tandem repeat (STR) analysis at any time after HSCT. Secondary graft failure was defined as a neutrophil count of 0.5×109 /L or less at 3 consecutive points after the confirmation of neutrophil engraftment.
Statistical considerations Differences between groups were examined using Fisher’s exact test for categorical variables and the Mann-Whitney U-test for continuous variables. Overall survival (OS) was calculated using the Kaplan-Meier method, and the log-rank test and post-hoc multicomparison test using the Holm method were performed to compare OS among groups classified according to the status of MC and SGF. Failure-free survival (FFS) was also calculated treating SGF or death from any cause as a failure. A log-rank test was also performed to evaluate the influence of factors for OS. To evaluate the influence of factors for the occurrence of MC and SGF, Fisher’s exact test and a logistic regression analysis were used for univariate and multivariate analyses, respectively. Factors with a P value of less than 0.10 in univariate analyses were subjected to multivariate analyses using the backward stepwise selection of covariates. Finally, P values of less than 0.05 were considered to be statistically significant. The
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Cochran-Armitage test was also performed to evaluate the influence of the dose of CY on the occurrence of MC and SGF. All statistical analyses were performed with EZR (Jichi Medical University Saitama Medical Center)[10], which is a graphical user interface for R (The R Foundation for Statistical Computing, version 3.4.0, Vienna, Austria).
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Results Patient characteristics In TRUMP, 819 patients were included in the additional survey. From among these, additional data were collected in 418 patients (51.0%), and these patients were ultimately included in this study. Their median age was 30 years (range 16 – 65 years) and in 95.6% of patients, CY was used in the conditioning regimens. In patients who received ATG or anti-lymphocyte globulin (ALG) in conditioning regimens, thymoglobulin was most frequently used with a median total dose and the most frequent dose of 5 mg/kg. In patients who received TBI or TLI in conditioning regimens, TBI was frequently used with a median total dose of 3 Gy and the most frequently dose of 2 Gy. The donor source was mainly bone marrow (87.3%). The median follow-up period for survivors was 1727 days. The characteristics of the patients are summarized in Table 1. We divided patients into 5 groups according to the occurrence of MC and/or SGF: patients who did not develop either MC or SGF (Group 0, n = 340, 81.3%), patients who developed MC that did not require either granulocyte-colony stimulating factor (G-CSF) or transfusion support (Group 1, n = 26, 6.2%), patients who developed MC (not SGF) that required G-CSF and/or transfusion support (Group 2, n = 16, 3.8%),
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patients who developed SGF with MC or complete recipient-type chimerism (Group 3, n = 19, 4.5%), and patients who developed SGF with complete donor-type chimerism (Group 4, n = 17, 4.1%). The sources of chimerism analyses at the onset of SGF were bone marrow (whole blood), peripheral blood (whole blood or T-cell compartment), both bone marrow and peripheral blood, or unknown in 8, 5, 5,1 patients, respectively in Group3, and 10, 4, 2,1 patients, respectively in Group 4. Overall, 61 patients (14.6%) developed MC (Groups 1, 2 and 3) and 36 (8.6%) developed SGF (Groups 3 and 4). OS rates in Group 0 (1year: 90.4%, 5 years: 83.5%) were not different from those in Group 1 (1 year: 100.0%, 5 years: 94.1%, P = 0.61) or Group 2 (1 year: 87.5%, 5 years: 60.9%, P = 0.61). In contrast, OS rates in Group 3 (1 year: 52.1 %, 5 years: 52.1 %, P < 0.001) and Group 4 (1 year: 82.4%, 5 years: 56.3%, P = 0.003) were significantly lower than those in Group 0. OS rates were not different between Group 3 and Group 4 (P = 0.61) (Figure 1). SGF developed later in Group 4 than in Group 3 (median days from transplantation: 99 days (range: 24 -212 days) vs. 55 days (range 24-238 days), P = 0.04). FFS rates in Group 0 were significantly higher than those in Group 3 ( P < 0.001) or Group 4 (P < 0.001). On the other hand, FFS rates were not different between Group 3 and Group 4 (P = 0.21).
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Risk factors for the development of secondary graft failure with mixed chimerism or complete recipient-type chimerism Risk factors for the development of SGF with MC or complete recipient-type chimerism were examined by comparing Group 3 with Group 0. In univariate analyses, the use of Flu, in vivo T-cell depletion (TCD), and the absence of TBI and total lymphoid irradiation (TLI) were significantly associated with the development of SGF with MC or complete recipient-type chimerism. The dose of CY was not significantly associated with this type of SGF, even with a trend analysis (Cochran-Armitage test, P = 0.103). In multivariate analyses, the use of Flu (odds ratio: 4.26, P = 0.03) and the absence of irradiation (odds ratio: 5.88, P < 0.001) in conditioning regimens were associated with the development of SGF with MC or complete recipient-type chimerism (Table 2). However, these factors alone were not significantly associated with OS (OS rates in 5 years: use of Flu vs. no use of Flu; 80.4% vs. 83.7%, P = 0.174, and use of irradiation vs no use of irradiation; 83.1% vs 77.8%, P = 0.411). In Group 3, 7 patients received ganciclovir as a pre-emptive therapy for cytomegalovirus (CMV) before the development of SGF, and 4 of them received it within a month before the development of SGF. Two patients had CMV disease despite
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the pre-emptive therapy.
Risk factors for the development of secondary graft failure with complete donor-type chimerism Risk factors for the development of SGF with complete donor-type chimerism were examined by comparing Group 4 with Group 0. In univariate analyses, donor type, the dose of CY, the use of Flu, and the use of in vivo TCD were associated with the development of SGF with complete donor-type chimerism, significantly or with borderline significance. However, none of these factors was significant in a multivariate analysis (Table 3(A)). When multivariate analyses were performed only for patients who received CY in conditioning regimens (n = 393), the use of Flu was significantly associated with SGF with complete donor-type chimerism (odds ratio: 8.68, P = 0.04) (Table 3(B)). Even in these patients, the use of Flu alone did not significantly influence OS (OS rates in 5 years: use of Flu vs. no use of Flu; 82.2% vs. 85.5%, P = 0.168). In group 4, 7 patients received ganciclovir or valgancyclovir as a pre-emptive therapy for CMV before the development of SGF, and 2 of them received it within a month before the development of SGF. One had CMV disease despite the pre-emptive
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therapy. In
addition, one
patient
had Epstein-Barr virus (EBV)-associated
lymphoproliferative disease before the development of SGF.
Clinical course of secondary graft failure We further investigated the clinical course of patients with SGF, with particular focus on the use of post-transplant immunosuppressants (IS). SGF developed before the tapering of IS was started in 14 of 19 patients in Group 3, including 2 patients in whom the tapering of IS was not started within a year, and in 9 of 17 patients in Group 4, including 5 patients in whom the tapering of IS was not started within a year. The median days when the tapering of IS started after HSCT were 70 and 55 in Groups 3 and 4, respectively. On the other hand, in Group 0, tapering of IS started at a median of 87 days after HSCT, and the tapering of IS was not started within 1 year in 88 patients (25.9%). After the onset of SGF, 2 patients experienced spontaneous hematological recovery at 66 and 152 days after HSCT in Group 3, and 2 patients did at 158 and 175 days after HSCT in Group 4. An increase in IS was effective for SGF in only one patient in Group 3, but in 5 patients in Group 4. A decrease or discontinuation of IS was effective in one patient in Group 3, but in none of the patients in Group 4. A second
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transplantation was successfully performed in 10 and 4 patients in Groups 3 and 4, respectively. On the other hand, 2 patients died early without hematological recovery, and 2 patients died with complication after the neutrophil engraftment in patients who underwent second transplantation in Group 3. In patient who underwent second
transplantation in Group 4, 3 patients died early without hematological recovery. In addition, hematological recovery was not observed in one patient who received only CD34+-selected peripheral blood cells (Table 4).
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Discussion A relatively high incidence of MC has been reported in patients who received HSCT for AA[11-14], and MC has been shown to be associated with both primary[11] and secondary graft failure[15, 16]. We focused on MC after neutrophil engraftment and SGF especially in recent years to clarify the influence of Flu in conditioning regimens. Umbilical cord blood (UCB) transplantation was excluded from this study. Although UCB transplantation for AA has recently been shown to give encouraging results in some studies[17-19], the conditioning regimens were different from those in HSCT using bone marrow or peripheral blood, since many patients in Japan did not use CY or ATG as pre-transplant conditioning for UCB transplantation[17, 18]. Therefore, it is difficult to analyze these patients together. In our current study, MC and SGF were observed in 14.6% and 8.6% of patients, respectively. The rate of MC was lower than those in previous studies[11-14]. These studies reported the results of serial chimerism analyses that were performed mainly as scheduled. On the other hand, in our study, chimerism analyses were performed according to the physician’s discretion or institutional policy. Chimerism analyses might not be performed in patients without severe cytopenia, and this could have led to the difference in the rate of MC compared to previous studies. Unlike the rate of MC, the rate of SGF was
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similar to those in previous studies[12-14, 20]. The occurrence of SGF is easily detected in daily practice, and would be expected to be correctly reported to the registry database used in our study. As in a previous study[12], OS rates in patients with MC but without SGF were not significantly different from those in patients without MC (Figure 1). Therefore, only patients who developed SGF were compared to those without MC or SGF in subsequent analyses. The use of Flu was significantly associated with the incidence of SGF with MC or complete recipient-type chimerism in our study, as well as the absence of irradiation, which is a well-known risk factor for primary graft failure[4, 21]. In conditioning regimens including Flu, the dose of CY was usually reduced, which suggests that not only the use of Flu but also a reduced dose of CY might influence the incidence of graft failure[4]. However, the dose of CY did not affect the incidence of SGF in our study, which is consistent with a previous study including only Japanese patients with AA who received HSCT from an HLA-matched sibling[22]. In the registry data, the range of CY doses in each group was relatively wide, and this might exclude the dose of CY as a risk factor for SGF. Similar to previous studies, patient age[23], donor type[20], donor source[24], and the type of graft-versus-host disease (GVHD) prophylaxis[12] did not affect the incidence of SGF.
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The use of Flu was also associated with SGF with complete donor-type chimerism, but only if patients who used CY in their conditioning regimens were included in the multivariate analyses. Yoshida et al. showed that the use of conditioning regimens including Flu was an independent risk factor for SGF with complete donor-type chimerism in HSCT for children with AA[6]. In addition, they demonstrated that a reduced CY dose was associated with SGF with complete donor-type chimerism in patients who used Flu in pre-transplant conditioning. However, the dose of CY was not a risk factor for SGF with complete donor-type chimerism in adult patients with AA in our study even when only patients who used Flu in pretransplant conditioning were included in the analysis (P = 0.508). Hama et al. reported that refractory cytopenia of childhood (RCC) and conditioning regimens using Flu and CY were risk factors for SGF with complete donor-type chimerism in HSCT for pediatric patients with acquired bone marrow failure, based on a review of bone marrow samples in a single institution[7]. SGF with complete donor-type chimerism in pediatric patients with AA may not be the same as that in adult patients with AA. Conditioning regimens using Flu while reducing the dose of CY might decrease the cytotoxic effect, and that might lead to the minimal residence of recipient-type lymphocytes, which disturb the graft function. On the other hand, Kong et al. reported
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the association between the microenviroment in bone marrow and SGF with complete donor-type chimerism[25]. Considering that only the use of Flu may affect the occurrence of SGF with both recipient- and donor-type chimerism, the effect of Flu and CY on microenviroment in bone marrow may be different, and this different effect may also vary with age. These risk factors for both types of SGF did not affect OS, probably because only some of the patients actually developed secondary SGF. The lower toxicity of regimens including Flu might offset the worse outcomes of SGF. The relatively long-term use of post-transplant IS is recommended to avoid not only GVHD but also late graft failure in patients with AA[26]. In our study, tapering of IS within a year was not significantly associated with an increase in the occurrence of SGF with MC or complete recipient-type chimerism (P = 1.0) or SGF with complete donor-type chimerism (P = 0.55). In patients with an early start of tapering of IS, donor-derived lymphocytes and the development of GVHD were augmented, and as a result, SGF might decrease in these patients. A decrease or discontinuation of IS was not effective in most of patients who developed SGF with both MC/complete recipient-type chimerism and complete donor-type chimerism. This result was compatible with previous reports[14, 26]. On
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the other hand, an increase in IS was effective, especially in patients who developed SGF with complete donor-type chimerism. In this type of SFG, residual recipient-type lymphocytes in peripheral blood might attack donor-derived hematopoiesis in bone marrow. An increase in IS might ameliorate this condition and led to normal hematopoiesis. Second transplantation led to the hematological recovery after SGF in 10 of 12 patients with MC/complete recipient-type chimerism and 4 of 8 patients with complete donor-type chimerism. However, some patients died with transplant-related complication, with or without hematological recovery. Infusion of CD34+ selected peripheral blood cells without conditioning demonstrated the promising results in some reports[27-30], and should be considered an alternative intervention for SGF. However, a patient who underwent this intervention did not achieve hematological recovery in our study. Our study had several limitations. First, the number of patients who actually developed SGF was small. Second, the timing and source of chimerism analyses varied according to each physician’s discretion, and serial results of chimerism analyses were not always obtained. As a result, it was difficult to interpret the mechanism of the response of SGF following interventions in some patients. However, even with these limitations, our study included a large number of adult AA patients with detailed
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results of chimerism analyses, and we believe that the results provide important insights to clarify the current state of MC and/or SGF after HSCT in adult patients with AA, especially after the introduction of new conditioning regimens including Flu. In conclusion, the occurrence of SGF with both MC/recipient-type and donor-type chimerism after HSCT for AA was associated with inferior OS, and the conditioning regimens influenced the occurrence of SGF. The use of Flu may increase the occurrence of SGF with both MC/recipient-type and donor-type chimerism. When SGF occurs, efficacious intervention may differ according to the type of SGF.
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Key Words aplastic anemia, allogeneic hematopoietic stem cell transplantation, mixed chimerism, secondary graft failure
Acknowledgement The authors thank all of the physicians and data managers who contributed valuable data on transplantation to TRUMP, especially those who participated in the additional survey. We also thank the staff of the Japanese Data Center for Hematopoietic Cell Transplantation (JDCHCT) for their assistance.
Conflict of interest disclosures Yamazaki H reports receiving honoraria from Sanofi K.K., and Kanda Y reports receiving research funding from Sanofi K.K. and Shionogi & CO., LTD. with regard to this study.
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chronic GVHD with peripheral blood progenitor cells than bone marrow in HLA-matched sibling donor transplants for young patients with severe acquired aplastic anemia. Blood 2007;110:1397-1400. 25.
Kong Y, Chang YJ, Wang YZ, et al. Association of an impaired bone marrow
microenvironment with secondary poor graft function after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013;19:1465-1473. 26.
Marsh JC, Ball SE, Cavenagh J, et al. Guidelines for the diagnosis and
management of aplastic anaemia. Br J Haematol 2009;147:43-70.
29
27.
Stasia A, Ghiso A, Galaverna F, et al. CD34 selected cells for the treatment of
poor graft function after allogeneic stem cell transplantation. Biol Blood Marrow
Transplant 2014;20:1440-1443. 28.
Askaa B, Fischer-Nielsen A, Vindelov L, Haastrup EK, Sengelov H. Treatment
of poor graft function after allogeneic hematopoietic cell transplantation with a booster of CD34-selected cells infused without conditioning. Bone Marrow Transplant 2014;49:720-721. 29.
Klyuchnikov E, El-Cheikh J, Sputtek A, et al. CD34(+)-selected stem cell boost
without further conditioning for poor graft function after allogeneic stem cell transplantation in patients with hematological malignancies. Biol Blood Marrow
Transplant 2014;20:382-386. 30.
Mainardi C, Ebinger M, Enkel S, et al. CD34(+) selected stem cell boosts can
improve poor graft function after paediatric allogeneic stem cell transplantation. Br J
Haematol 2018;180:90-99.
30
Figure legends
Figure 1 Overall survival according to the occurrence of mixed chimerism and/or secondary graft failure
Overall survival (OS) rates in Group 0 were not different from those in Group 1 or Group 2. In contrast, OS rates in Group 3 and Group 4 were significantly lower than those in Group 0. OS rates were not different between Group 3 and Group 4.
31
Table 1 Patient characteristics Group 0 (n = 340)
Group 1 (n = 26)
Group 2 (n = 16)
Group 3 (n = 19)
Group 4 (n = 17)
30 (16-65) 175/165
25 (16-56) 13/13
27 (19-55) 11/5
34 (17-56) 11/8
27 (16-51) 10/7
65/145/70
10/9/5
1/9/4
1/13/4
2/9/6
148/112
13/10
9/5
12/4
11/4
715
404
574
840
854
293/47
25/1
15/1
15/4
17/0
146/13/6 /59/7/24/47
15/0/0 /5/1/0/3
11/0/0 /3/0/1/0
6/0/0 /2/0/3/6
2/0/1 /6/1/1/4
74/259
5/21
3/13
4/15
2/15
163/53/57/27
10/7/6/1
9/1/4/0
13/2/2/1
9/5/3/0
+/+/-
317/16 94/116/87 175/143 249/84 192/148
26/0 4/13/6 15/11 9/17 23/3
15/1 3/4/7 6/10 7/9 13/3
18/1 9/6/3 15/3 7/12 19/0
17/0 5/10/1 13/2 13/4 15/2
CSA/CSA+MTX/TAC /TAC+MTX/Others
16/169/10 /133/5
2/18/2 /4/0
0/11/0 /5/0
1/8/1 /9/0
0/5/0 /12/0
Age median(range) Sex Male/Female Severity at HSCT less than SAA/SAA/VSAA IST* before HSCT +/Duration from Diagnosis to HSCT median days Donor source BM/PB Donor type MRD/1MM-RD/23MM-RD /MUD88** /MUD66**/MUD*/MUD87
D-R sex combination Female to Male /Others
ABO compatibility Match/Min/Maj/Maj & Min +/low/middle/high** +/-
CY use CY dose Flu use TBI/TLI use In vivo TCD*** GVHD prophylaxis
*Only treatments including anti-thymocyte globulin (ATG) or anti-lymphocyte globulin (ALG) were included in immunosuppresive therapies (IST). ** Matched unrelated donor 88 (MUD88) included donors with human leukocyte antigen (HLA) -A, -B, -C, -DRB1 8/8 allele-match, and MUD66 included donors with HLA -A, -B, -DRB1 6/6 allele-match. MUD included donors with HLA -A, -B, -DR 6/6 antigen-match, and MUD88, MUD66, or one allele-mismatched in HLA-A, -B, -C, -DRB1 unrelated donor (MUD87) were excluded. ***The dose of cyclophosphamide (CY) was classified as follows; low: less than 100 mg/kg, middle; 100 mg/kg or more than 100 mg/kg, but less than 200 mg/kg, high; 200 mg/kg or more than 200 mg/kg. **** In vivo T-cell depletion included the use of ATG, ALG, or alemtuzumab. Abbreviations: HSCT; hematopoietic stem cell transplantation, SAA; severe aplastic anemia, VSAA; very severe aplastic anemia, BM; bone marrow, PB; peripheral blood, MRD; matched related donor, 1MM-RD; HLA one antigen-mismatched in graft-versus-host direction related donor, 1MM-RD; HLA 2-3 antigen-mismatched in graft-versus-host direction related donor, D-R; donor-recipient, Min.; minor mismatch, Maj.; major mismatch, Flu; fludarabine, TBI; total body irradiation, TLI; total lymphoid irradiation, TCD; T-cell 32
depletion, GVHD; graft-versus-host disease, CSA; cyclosporine, MTX; methotrexate, TAC; tacrolimus Table 2 Risk factors for secondary graft failure with mixed chimerism or complete recipient-type chimerism
Univariate analysis P value
Multivariate analysis Odds ratio
P value
(95% C.I.) Flu use
+/-
Group 0
175/143
Group 3 TBI/TLI use
+/-
Group 0
249/84
+/-
Group 0
0.001
Group 3
0.17 (0.06 – 0.48)
7/12 177/158
4.26
0.002
17/2
Abbreviations: Flu; fludarabine, TBI; total body irradiation, TLI; total lymphoid irradiation, TCD; T-cell depletion, C.I.; confidence interval
33
0.026
(1.19 – 15.30)
15/3
Group 3 In vivo TCD
0.026
< 0.001
Table 3
Risk factors for the development of secondary graft failure with complete
donor-type chimerism (A) All Patients Univariate analysis***
P value Donor type Group 0
146/13/6/59/7/24/47
/MUD88* /MUD66*/MUD*/MUD87
Group 4
2/0/1/6/1/1/4
CY dose low/middle/high**
Group 0
94/116/87
MRD/1MM-RD/23MM-RD
0.027
0.069
Group 4 Flu use
+/-
Group 0 Group 4
In vivo TCD +/-
Group 0 Group 4
5/10/1 175/143 0.016 13/2 177/158 0.079 13/4
* Matched unrelated donor 88 (MUD88) included donors with human leukocyte antigen (HLA) -A, -B, -C, -DRB1 8/8 allele-match, and MUD66 included donors with HLA -A, -B, -DRB1 6/6 allele-match. MUD included donors with HLA -A, -B, -DR 6/6 antigen-match, and MUD88, MUD66, or one allele-mismatched in HLA-A, -B, -C, -DRB1 unrelated donor (MUD87) were excluded. ** The dose of cyclophosphamide (CY) was classified as follows; low: less than 100 mg/kg, middle; 100 mg/kg or more than 100 mg/kg, but less than 200 mg/kg, high; 200 mg/kg or more than 200 mg/kg. *** Significant factor was not detected by a multivariate analysis. Abbreviations: MRD; matched related donor, 1MM-RD; HLA one antigen-mismatched in graft-versus-host direction related donor, 1MM-RD; HLA 2-3 antigen-mismatched in graft-versus-host direction related donor, Flu; fludarabine, TCD; T-cell depletion
34
(B) Patients who received cyclophosphamide in conditioning regimens
Univariate analysis P value
Multivariate analysis Odds ratio
P value
(95% C.I.) Donor type MRD/1MM-RD/23MM-RD /MUD88*/MUD66*/MUD*/MUD87
CY dose
Flu use
low/middle/high**
+/－
In vivo TCD
+/－
Group 0
140/13/2/54/7/23/45
Group 4
2/0/1/6/1/1/4
Group 0
78/116/87
Group 4
5/10/1
Group 0
157/142
Group 4
13/2
Group 0
172/144
Group 4
13/4
0.012
0.069
0.014
8.68 (1.11 - 67.80)
0.084
* Matched unrelated donor 88 (MUD88) included donors with human leukocyte antigen (HLA) -A, -B, -C, -DRB1 8/8 allele-match, and MUD66 included donors with HLA -A, -B, -DRB1 6/6 allele-match. MUD included donors with HLA -A, -B, -DR 6/6 antigen-match, and MUD88, MUD66, or one allele-mismatched in HLA-A, -B, -C, -DRB1 unrelated donor (MUD87) were excluded. ** The dose of cyclophosphamide (CY) was classified as follows; low: less than 100 mg/kg, middle; 100 mg/kg or more than 100 mg/kg, but less than 200 mg/kg, high; 200 mg/kg or more than 200 mg/kg. Abbreviations: MRD; matched related donor, 1MM-RD; HLA one antigen-mismatched in graft-versus-host direction related donor, 1MM-RD; HLA 2-3 antigen-mismatched in graft-versus-host direction related donor, Flu; fludarabine, TCD; T-cell depletion
35
0.039
Table 4 Outcomes of interventions for secondary graft failure
Group 3
Group 4
No. of Patients (effective)
No. of Patients (effective)
Observation
5 ( 2)
3 (2)
Increase in IS
3 (1)
7 (5)
Decrease in IS
8 (1)
3 (0)
DLI
2 (0)
1 (0)
12 (10)
8 (4)
Second transplantation
Abbreviations: IS; immunosuppressant, DLI; donor lymphocyte infusion
36
Journal Pre-proof
Mixed chimerism and secondary graft failure in allogeneic hematopoietic stem cell transplantation for aplastic anemia Shinichi Kako M.D. , Hirohito Yamazaki M.D. , Kazuteru Ohashi M.D. , Yukiyasu Ozawa M.D. , Shuichi Ota M.D. , Yoshinobu Kanda M.D. , Tetsuo Maeda M.D. , Jun Kato M.D. , Ken Ishiyama M.D. , Ken-ichi Matsuoka M.D. , Toshihiro Miyamoto M.D. , Hiroatsu Iida M.D. , Kazuhiro Ikegame M.D. , Takahiro Fukuda M.D. , Tatsuo Ichinohe M.D. , Yoshiko Atsuta M.D. , Takehiko Mori M.D. , on behalf of the Adult Aplastic Anemia Working Group of the Japanese Society for Hematopoietic Cell Transplantation PII: DOI: Reference:
S1083-8791(19)30660-3 https://doi.org/10.1016/j.bbmt.2019.10.004 YBBMT 55752
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
6 August 2019 1 October 2019
Please cite this article as: Shinichi Kako M.D. , Hirohito Yamazaki M.D. , Kazuteru Ohashi M.D. , Yukiyasu Ozawa M.D. , Shuichi Ota M.D. , Yoshinobu Kanda M.D. , Tetsuo Maeda M.D. , Jun Kato M.D. , Ken Ishiyama M.D. , Ken-ichi Matsuoka M.D. , Toshihiro Miyamoto M.D. , Hiroatsu Iida M.D. , Kazuhiro Ikegame M.D. , Takahiro Fukuda M.D. , Tatsuo Ichinohe M.D. , Yoshiko Atsuta M.D. , Takehiko Mori M.D. , on behalf of the Adult Aplastic Anemia Working Group of the Japanese Society for Hematopoietic Cell Transplantation, Mixed chimerism and secondary graft failure in allogeneic hematopoietic stem cell transplantation for aplastic anemia, Biology of Blood and Marrow Transplantation (2019), doi: https://doi.org/10.1016/j.bbmt.2019.10.004
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Society for Transplantation and Cellular Therapy
Highlights
The influence of MC and/or SGF in HSCT for AA was retrospectively evaluated.
SGF with both MC/recipient- and donor-type chimerism was observed.
Patients who developed SGF with both types of chimerism had unfavorable outcomes.
The use of fludarabine may affect the occurrence of SGF with both types of chimerism.
1
Regular Manuscript
Mixed chimerism and secondary graft failure in allogeneic hematopoietic stem cell transplantation for aplastic anemia
Shinichi Kako, M.D.1, Hirohito Yamazaki, M.D.2, Kazuteru Ohashi, M.D.3, Yukiyasu Ozawa, M.D.4, Shuichi Ota, M.D.5, Yoshinobu Kanda, M.D.1, Tetsuo Maeda, M.D.6, Jun Kato, M.D.7, Ken Ishiyama, M.D.8, Ken-ichi Matsuoka, M.D.9, Toshihiro Miyamoto, M.D.10, Hiroatsu Iida, M.D.11, Kazuhiro Ikegame, M.D.12, Takahiro Fukuda, M.D.13, Tatsuo Ichinohe, M.D.14, Yoshiko Atsuta, M.D.15, Takehiko Mori, M.D.7, on behalf of the Adult Aplastic Anemia Working Group of the Japanese Society for Hematopoietic Cell Transplantation
1Division
of Hematology, Jichi Medical University Saitama Medical Center, Saitama,
Japan, 2Division of Transfusion Medicine, Kanazawa University Hospital, Kanazawa, Japan, 3Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan, 4Department of Hematology, Japanese Red
2
Cross Nagoya First Hospital, Nagoya, Japan, 5Department of Hematology, Sapporo Hokuyu Hospital, Sapporo, Japan, 6Department of Hematology and Oncology, Osaka University Hospital, Osaka, Japan, 7Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan,
8Department
of Hematology,
Kanazawa University Hospital, Kanazawa, Japan, 9Division of Hematology/Oncology, Okayama University Hospital, Hiroshima, Japan,
10Medicine
and Biosystemic Science,
Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan,
11Division
of Cell Therapy, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
12Division
of Hematology, Department of Internal Medicine, Hyogo College of
Medicine, Hyogo, Japan 13Hematopoietic Stem Cell Transplantation Division, National Cancer Center Hospital, Tokyo, Japan
14Department
of Hematology and Oncology,
Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan,
15Japanese
Data Center for Hematopoietic Cell Transplantation,
Nagoya, Japan
Corresponding author: Shinichi Kako, M.D. Division of Hematology, Department of Internal Medicine, Jichi Medical University Saitama Medical Center
3
1-847 Amanuma, Omiya-ku, Saitama-city, Saitama 330-8503, Japan TEL: +81-48-647-2111 ext. 5881 FAX: +81-48-648-5188 E-mail: [email protected]
Short title Secondary graft failure in transplant for aplastic anemia
Conflict of interest disclosures Yamazaki H reports receiving honoraria from Sanofi K.K., and Kanda Y reports receiving research funding from Sanofi K.K. and Shionogi & CO., LTD. with regard to this study.
4
Abstract Mixed chimerism (MC) and/or secondary graft failure (SGF) with recipient- or donor-type chimerism is a major obstacle in allogeneic transplantation for aplastic anemia (AA). From a registry database in Japan, patients with AA aged more than 15 years who underwent a first allogeneic bone marrow or peripheral blood transplantation between 2000 and 2014 and achieved engraftment were included in this study. MC that did not require either granulocyte-colony stimulating factor (G-CSF) or transfusion support (Group 1), MC (not SGF) that required G-CSF and/or transfusion support (Group 2), SGF with MC or complete recipient-type chimerism (Group 3), and SGF with complete donor-type chimerism (Group 4) developed in 26, 16, 19, and 17 patients, respectively. The overall median follow-up period for survivors was 1727 days. The overall survival rate (OS) was 90.4% at 1 year and 83.5% at 5 years in patients without MC or SGF (n = 340), which was not different from OS in Group 1 or 2. However, inferior OS was observed in Group 3 (1 year: 52.1%, 5 years: 52.1%) and Group 4 (1 year: 82.4%, 5 years: 56.3%). In multivariate analyses, the use of fludarabine (Flu) and the absence of irradiation in conditioning were associated with the development of SGF with MC or complete recipient-type chimerism, and the use of Flu in conditioning was associated with SGF with complete donor-type chimerism. In
5
conclusion, the use of Flu may affect the occurrence of SGF with both recipient- and donor-type chimerism.
6
Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective and curative treatment option for patients with aplastic anemia (AA). High-dose cyclophosphamide (CY) (200 mg/kg) combined with anti-thymocyte globulin (ATG) is frequently used as a conditioning regimen, and shows excellent outcomes especially in HSCT from a human leukocyte antigen (HLA)-matched sibling[1]. However, the cardiotoxicity of high-dose CY is a major problem[2], since patients with AA often have cardiac dysfunction due to long-term anemia and iron overload associated with massive transfusion. Recently, conditioning regimens involving fludarabine (Flu) and reduced-dose CY are increasingly being used to avoid the toxicity of high-dose CY. Good clinical courses without graft rejection in HSCT from an HLA-matched sibling have been reported[3], and even in HSCT from alternative donors, a conditioning regimen that includes Flu and reduced-dose CY can overcome graft rejection with the addition of low-dose total body irradiation (TBI)[4]. However, mixed chimerism (MC) after neutrophil engraftment and/or secondary graft failure (SGF) is still observed with such conditioning regimens, especially in HSCT from an HLA-matched sibling[5]. Moreover, SGF with complete donor-type chimerism appears to be another obstacle. In Japan, this condition has mainly been reported in pediatric patients with AA [6, 7], but
7
has also been observed in adult patients with AA[5]. However, the risk factors for MC and/or SGF with recipient- or donor-type chimerism and the influence of these conditions on the prognosis remain to be elucidated, especially in adult patients with AA. Therefore, we performed a retrospective analysis using the registry database of the Japanese Society for Hematopoietic Cell Transplantation (JSHCT) to clarify the current state of MC and/or SGF after HSCT in adult patients with AA.
8
Patients and Methods Data source The registry database of JSCHT is called the Transplant Registry Unified Management Program (TRUMP)[8, 9]. In TRUMP, patients with AA aged more than 15 years who underwent a first allogeneic bone marrow or peripheral blood transplantation between 2000 and 2014 and achieved neutrophil engraftment were initially selected. Next, an additional survey was conducted for these patients to collect detailed data regarding MC and SGF, and patients for whom this information was available were included in this study. This study was planned by the Adult Aplastic Anemia Working Group of JSHCT and approved by the data management committee of TRUMP and the institutional review board of Jichi Medical University Saitama Medical Center.
Definition of mixed chimerism and secondary graft failure Chimerism analyses were performed using peripheral blood or bone marrow cells. The timing and source of chimerism analyses depended on each physician’s discretion. MC was defined as the detection of at least one recipient-type cell in sex-chromosome analysis, or a rate of recipient-type cells of at least 5% in
9
sex-chromosome fluorescence in situ hybridization (FISH) analysis or short tandem repeat (STR) analysis at any time after HSCT. Secondary graft failure was defined as a neutrophil count of 0.5×109 /L or less at 3 consecutive points after the confirmation of neutrophil engraftment.
Statistical considerations Differences between groups were examined using Fisher’s exact test for categorical variables and the Mann-Whitney U-test for continuous variables. Overall survival (OS) was calculated using the Kaplan-Meier method, and the log-rank test and post-hoc multicomparison test using the Holm method were performed to compare OS among groups classified according to the status of MC and SGF. Failure-free survival (FFS) was also calculated treating SGF or death from any cause as a failure. A log-rank test was also performed to evaluate the influence of factors for OS. To evaluate the influence of factors for the occurrence of MC and SGF, Fisher’s exact test and a logistic regression analysis were used for univariate and multivariate analyses, respectively. Factors with a P value of less than 0.10 in univariate analyses were subjected to multivariate analyses using the backward stepwise selection of covariates. Finally, P values of less than 0.05 were considered to be statistically significant. The
10
Cochran-Armitage test was also performed to evaluate the influence of the dose of CY on the occurrence of MC and SGF. All statistical analyses were performed with EZR (Jichi Medical University Saitama Medical Center)[10], which is a graphical user interface for R (The R Foundation for Statistical Computing, version 3.4.0, Vienna, Austria).
11
Results Patient characteristics In TRUMP, 819 patients were included in the additional survey. From among these, additional data were collected in 418 patients (51.0%), and these patients were ultimately included in this study. Their median age was 30 years (range 16 – 65 years) and in 95.6% of patients, CY was used in the conditioning regimens. In patients who received ATG or anti-lymphocyte globulin (ALG) in conditioning regimens, thymoglobulin was most frequently used with a median total dose and the most frequent dose of 5 mg/kg. In patients who received TBI or TLI in conditioning regimens, TBI was frequently used with a median total dose of 3 Gy and the most frequently dose of 2 Gy. The donor source was mainly bone marrow (87.3%). The median follow-up period for survivors was 1727 days. The characteristics of the patients are summarized in Table 1. We divided patients into 5 groups according to the occurrence of MC and/or SGF: patients who did not develop either MC or SGF (Group 0, n = 340, 81.3%), patients who developed MC that did not require either granulocyte-colony stimulating factor (G-CSF) or transfusion support (Group 1, n = 26, 6.2%), patients who developed MC (not SGF) that required G-CSF and/or transfusion support (Group 2, n = 16, 3.8%),
12
patients who developed SGF with MC or complete recipient-type chimerism (Group 3, n = 19, 4.5%), and patients who developed SGF with complete donor-type chimerism (Group 4, n = 17, 4.1%). The sources of chimerism analyses at the onset of SGF were bone marrow (whole blood), peripheral blood (whole blood or T-cell compartment), both bone marrow and peripheral blood, or unknown in 8, 5, 5,1 patients, respectively in Group3, and 10, 4, 2,1 patients, respectively in Group 4. Overall, 61 patients (14.6%) developed MC (Groups 1, 2 and 3) and 36 (8.6%) developed SGF (Groups 3 and 4). OS rates in Group 0 (1year: 90.4%, 5 years: 83.5%) were not different from those in Group 1 (1 year: 100.0%, 5 years: 94.1%, P = 0.61) or Group 2 (1 year: 87.5%, 5 years: 60.9%, P = 0.61). In contrast, OS rates in Group 3 (1 year: 52.1 %, 5 years: 52.1 %, P < 0.001) and Group 4 (1 year: 82.4%, 5 years: 56.3%, P = 0.003) were significantly lower than those in Group 0. OS rates were not different between Group 3 and Group 4 (P = 0.61) (Figure 1). SGF developed later in Group 4 than in Group 3 (median days from transplantation: 99 days (range: 24 -212 days) vs. 55 days (range 24-238 days), P = 0.04). FFS rates in Group 0 were significantly higher than those in Group 3 ( P < 0.001) or Group 4 (P < 0.001). On the other hand, FFS rates were not different between Group 3 and Group 4 (P = 0.21).
13
Risk factors for the development of secondary graft failure with mixed chimerism or complete recipient-type chimerism Risk factors for the development of SGF with MC or complete recipient-type chimerism were examined by comparing Group 3 with Group 0. In univariate analyses, the use of Flu, in vivo T-cell depletion (TCD), and the absence of TBI and total lymphoid irradiation (TLI) were significantly associated with the development of SGF with MC or complete recipient-type chimerism. The dose of CY was not significantly associated with this type of SGF, even with a trend analysis (Cochran-Armitage test, P = 0.103). In multivariate analyses, the use of Flu (odds ratio: 4.26, P = 0.03) and the absence of irradiation (odds ratio: 5.88, P < 0.001) in conditioning regimens were associated with the development of SGF with MC or complete recipient-type chimerism (Table 2). However, these factors alone were not significantly associated with OS (OS rates in 5 years: use of Flu vs. no use of Flu; 80.4% vs. 83.7%, P = 0.174, and use of irradiation vs no use of irradiation; 83.1% vs 77.8%, P = 0.411). In Group 3, 7 patients received ganciclovir as a pre-emptive therapy for cytomegalovirus (CMV) before the development of SGF, and 4 of them received it within a month before the development of SGF. Two patients had CMV disease despite
14
the pre-emptive therapy.
Risk factors for the development of secondary graft failure with complete donor-type chimerism Risk factors for the development of SGF with complete donor-type chimerism were examined by comparing Group 4 with Group 0. In univariate analyses, donor type, the dose of CY, the use of Flu, and the use of in vivo TCD were associated with the development of SGF with complete donor-type chimerism, significantly or with borderline significance. However, none of these factors was significant in a multivariate analysis (Table 3(A)). When multivariate analyses were performed only for patients who received CY in conditioning regimens (n = 393), the use of Flu was significantly associated with SGF with complete donor-type chimerism (odds ratio: 8.68, P = 0.04) (Table 3(B)). Even in these patients, the use of Flu alone did not significantly influence OS (OS rates in 5 years: use of Flu vs. no use of Flu; 82.2% vs. 85.5%, P = 0.168). In group 4, 7 patients received ganciclovir or valgancyclovir as a pre-emptive therapy for CMV before the development of SGF, and 2 of them received it within a month before the development of SGF. One had CMV disease despite the pre-emptive
15
therapy. In
addition, one
patient
had Epstein-Barr virus (EBV)-associated
lymphoproliferative disease before the development of SGF.
Clinical course of secondary graft failure We further investigated the clinical course of patients with SGF, with particular focus on the use of post-transplant immunosuppressants (IS). SGF developed before the tapering of IS was started in 14 of 19 patients in Group 3, including 2 patients in whom the tapering of IS was not started within a year, and in 9 of 17 patients in Group 4, including 5 patients in whom the tapering of IS was not started within a year. The median days when the tapering of IS started after HSCT were 70 and 55 in Groups 3 and 4, respectively. On the other hand, in Group 0, tapering of IS started at a median of 87 days after HSCT, and the tapering of IS was not started within 1 year in 88 patients (25.9%). After the onset of SGF, 2 patients experienced spontaneous hematological recovery at 66 and 152 days after HSCT in Group 3, and 2 patients did at 158 and 175 days after HSCT in Group 4. An increase in IS was effective for SGF in only one patient in Group 3, but in 5 patients in Group 4. A decrease or discontinuation of IS was effective in one patient in Group 3, but in none of the patients in Group 4. A second
16
transplantation was successfully performed in 10 and 4 patients in Groups 3 and 4, respectively. On the other hand, 2 patients died early without hematological recovery, and 2 patients died with complication after the neutrophil engraftment in patients who underwent second transplantation in Group 3. In patient who underwent second
transplantation in Group 4, 3 patients died early without hematological recovery. In addition, hematological recovery was not observed in one patient who received only CD34+-selected peripheral blood cells (Table 4).
17
Discussion A relatively high incidence of MC has been reported in patients who received HSCT for AA[11-14], and MC has been shown to be associated with both primary[11] and secondary graft failure[15, 16]. We focused on MC after neutrophil engraftment and SGF especially in recent years to clarify the influence of Flu in conditioning regimens. Umbilical cord blood (UCB) transplantation was excluded from this study. Although UCB transplantation for AA has recently been shown to give encouraging results in some studies[17-19], the conditioning regimens were different from those in HSCT using bone marrow or peripheral blood, since many patients in Japan did not use CY or ATG as pre-transplant conditioning for UCB transplantation[17, 18]. Therefore, it is difficult to analyze these patients together. In our current study, MC and SGF were observed in 14.6% and 8.6% of patients, respectively. The rate of MC was lower than those in previous studies[11-14]. These studies reported the results of serial chimerism analyses that were performed mainly as scheduled. On the other hand, in our study, chimerism analyses were performed according to the physician’s discretion or institutional policy. Chimerism analyses might not be performed in patients without severe cytopenia, and this could have led to the difference in the rate of MC compared to previous studies. Unlike the rate of MC, the rate of SGF was
18
similar to those in previous studies[12-14, 20]. The occurrence of SGF is easily detected in daily practice, and would be expected to be correctly reported to the registry database used in our study. As in a previous study[12], OS rates in patients with MC but without SGF were not significantly different from those in patients without MC (Figure 1). Therefore, only patients who developed SGF were compared to those without MC or SGF in subsequent analyses. The use of Flu was significantly associated with the incidence of SGF with MC or complete recipient-type chimerism in our study, as well as the absence of irradiation, which is a well-known risk factor for primary graft failure[4, 21]. In conditioning regimens including Flu, the dose of CY was usually reduced, which suggests that not only the use of Flu but also a reduced dose of CY might influence the incidence of graft failure[4]. However, the dose of CY did not affect the incidence of SGF in our study, which is consistent with a previous study including only Japanese patients with AA who received HSCT from an HLA-matched sibling[22]. In the registry data, the range of CY doses in each group was relatively wide, and this might exclude the dose of CY as a risk factor for SGF. Similar to previous studies, patient age[23], donor type[20], donor source[24], and the type of graft-versus-host disease (GVHD) prophylaxis[12] did not affect the incidence of SGF.
19
The use of Flu was also associated with SGF with complete donor-type chimerism, but only if patients who used CY in their conditioning regimens were included in the multivariate analyses. Yoshida et al. showed that the use of conditioning regimens including Flu was an independent risk factor for SGF with complete donor-type chimerism in HSCT for children with AA[6]. In addition, they demonstrated that a reduced CY dose was associated with SGF with complete donor-type chimerism in patients who used Flu in pre-transplant conditioning. However, the dose of CY was not a risk factor for SGF with complete donor-type chimerism in adult patients with AA in our study even when only patients who used Flu in pretransplant conditioning were included in the analysis (P = 0.508). Hama et al. reported that refractory cytopenia of childhood (RCC) and conditioning regimens using Flu and CY were risk factors for SGF with complete donor-type chimerism in HSCT for pediatric patients with acquired bone marrow failure, based on a review of bone marrow samples in a single institution[7]. SGF with complete donor-type chimerism in pediatric patients with AA may not be the same as that in adult patients with AA. Conditioning regimens using Flu while reducing the dose of CY might decrease the cytotoxic effect, and that might lead to the minimal residence of recipient-type lymphocytes, which disturb the graft function. On the other hand, Kong et al. reported
20
the association between the microenviroment in bone marrow and SGF with complete donor-type chimerism[25]. Considering that only the use of Flu may affect the occurrence of SGF with both recipient- and donor-type chimerism, the effect of Flu and CY on microenviroment in bone marrow may be different, and this different effect may also vary with age. These risk factors for both types of SGF did not affect OS, probably because only some of the patients actually developed secondary SGF. The lower toxicity of regimens including Flu might offset the worse outcomes of SGF. The relatively long-term use of post-transplant IS is recommended to avoid not only GVHD but also late graft failure in patients with AA[26]. In our study, tapering of IS within a year was not significantly associated with an increase in the occurrence of SGF with MC or complete recipient-type chimerism (P = 1.0) or SGF with complete donor-type chimerism (P = 0.55). In patients with an early start of tapering of IS, donor-derived lymphocytes and the development of GVHD were augmented, and as a result, SGF might decrease in these patients. A decrease or discontinuation of IS was not effective in most of patients who developed SGF with both MC/complete recipient-type chimerism and complete donor-type chimerism. This result was compatible with previous reports[14, 26]. On
21
the other hand, an increase in IS was effective, especially in patients who developed SGF with complete donor-type chimerism. In this type of SFG, residual recipient-type lymphocytes in peripheral blood might attack donor-derived hematopoiesis in bone marrow. An increase in IS might ameliorate this condition and led to normal hematopoiesis. Second transplantation led to the hematological recovery after SGF in 10 of 12 patients with MC/complete recipient-type chimerism and 4 of 8 patients with complete donor-type chimerism. However, some patients died with transplant-related complication, with or without hematological recovery. Infusion of CD34+ selected peripheral blood cells without conditioning demonstrated the promising results in some reports[27-30], and should be considered an alternative intervention for SGF. However, a patient who underwent this intervention did not achieve hematological recovery in our study. Our study had several limitations. First, the number of patients who actually developed SGF was small. Second, the timing and source of chimerism analyses varied according to each physician’s discretion, and serial results of chimerism analyses were not always obtained. As a result, it was difficult to interpret the mechanism of the response of SGF following interventions in some patients. However, even with these limitations, our study included a large number of adult AA patients with detailed
22
results of chimerism analyses, and we believe that the results provide important insights to clarify the current state of MC and/or SGF after HSCT in adult patients with AA, especially after the introduction of new conditioning regimens including Flu. In conclusion, the occurrence of SGF with both MC/recipient-type and donor-type chimerism after HSCT for AA was associated with inferior OS, and the conditioning regimens influenced the occurrence of SGF. The use of Flu may increase the occurrence of SGF with both MC/recipient-type and donor-type chimerism. When SGF occurs, efficacious intervention may differ according to the type of SGF.
23
Key Words aplastic anemia, allogeneic hematopoietic stem cell transplantation, mixed chimerism, secondary graft failure
Acknowledgement The authors thank all of the physicians and data managers who contributed valuable data on transplantation to TRUMP, especially those who participated in the additional survey. We also thank the staff of the Japanese Data Center for Hematopoietic Cell Transplantation (JDCHCT) for their assistance.
Conflict of interest disclosures Yamazaki H reports receiving honoraria from Sanofi K.K., and Kanda Y reports receiving research funding from Sanofi K.K. and Shionogi & CO., LTD. with regard to this study.
24
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30
Figure legends
Figure 1 Overall survival according to the occurrence of mixed chimerism and/or secondary graft failure
Overall survival (OS) rates in Group 0 were not different from those in Group 1 or Group 2. In contrast, OS rates in Group 3 and Group 4 were significantly lower than those in Group 0. OS rates were not different between Group 3 and Group 4.
31
Table 1 Patient characteristics Group 0 (n = 340)
Group 1 (n = 26)
Group 2 (n = 16)
Group 3 (n = 19)
Group 4 (n = 17)
30 (16-65) 175/165
25 (16-56) 13/13
27 (19-55) 11/5
34 (17-56) 11/8
27 (16-51) 10/7
65/145/70
10/9/5
1/9/4
1/13/4
2/9/6
148/112
13/10
9/5
12/4
11/4
715
404
574
840
854
293/47
25/1
15/1
15/4
17/0
146/13/6 /59/7/24/47
15/0/0 /5/1/0/3
11/0/0 /3/0/1/0
6/0/0 /2/0/3/6
2/0/1 /6/1/1/4
74/259
5/21
3/13
4/15
2/15
163/53/57/27
10/7/6/1
9/1/4/0
13/2/2/1
9/5/3/0
+/+/-
317/16 94/116/87 175/143 249/84 192/148
26/0 4/13/6 15/11 9/17 23/3
15/1 3/4/7 6/10 7/9 13/3
18/1 9/6/3 15/3 7/12 19/0
17/0 5/10/1 13/2 13/4 15/2
CSA/CSA+MTX/TAC /TAC+MTX/Others
16/169/10 /133/5
2/18/2 /4/0
0/11/0 /5/0
1/8/1 /9/0
0/5/0 /12/0
Age median(range) Sex Male/Female Severity at HSCT less than SAA/SAA/VSAA IST* before HSCT +/Duration from Diagnosis to HSCT median days Donor source BM/PB Donor type MRD/1MM-RD/23MM-RD /MUD88** /MUD66**/MUD*/MUD87
D-R sex combination Female to Male /Others
ABO compatibility Match/Min/Maj/Maj & Min +/low/middle/high** +/-
CY use CY dose Flu use TBI/TLI use In vivo TCD*** GVHD prophylaxis
*Only treatments including anti-thymocyte globulin (ATG) or anti-lymphocyte globulin (ALG) were included in immunosuppresive therapies (IST). ** Matched unrelated donor 88 (MUD88) included donors with human leukocyte antigen (HLA) -A, -B, -C, -DRB1 8/8 allele-match, and MUD66 included donors with HLA -A, -B, -DRB1 6/6 allele-match. MUD included donors with HLA -A, -B, -DR 6/6 antigen-match, and MUD88, MUD66, or one allele-mismatched in HLA-A, -B, -C, -DRB1 unrelated donor (MUD87) were excluded. ***The dose of cyclophosphamide (CY) was classified as follows; low: less than 100 mg/kg, middle; 100 mg/kg or more than 100 mg/kg, but less than 200 mg/kg, high; 200 mg/kg or more than 200 mg/kg. **** In vivo T-cell depletion included the use of ATG, ALG, or alemtuzumab. Abbreviations: HSCT; hematopoietic stem cell transplantation, SAA; severe aplastic anemia, VSAA; very severe aplastic anemia, BM; bone marrow, PB; peripheral blood, MRD; matched related donor, 1MM-RD; HLA one antigen-mismatched in graft-versus-host direction related donor, 1MM-RD; HLA 2-3 antigen-mismatched in graft-versus-host direction related donor, D-R; donor-recipient, Min.; minor mismatch, Maj.; major mismatch, Flu; fludarabine, TBI; total body irradiation, TLI; total lymphoid irradiation, TCD; T-cell 32
depletion, GVHD; graft-versus-host disease, CSA; cyclosporine, MTX; methotrexate, TAC; tacrolimus Table 2 Risk factors for secondary graft failure with mixed chimerism or complete recipient-type chimerism
Univariate analysis P value
Multivariate analysis Odds ratio
P value
(95% C.I.) Flu use
+/-
Group 0
175/143
Group 3 TBI/TLI use
+/-
Group 0
249/84
+/-
Group 0
0.001
Group 3
0.17 (0.06 – 0.48)
7/12 177/158
4.26
0.002
17/2
Abbreviations: Flu; fludarabine, TBI; total body irradiation, TLI; total lymphoid irradiation, TCD; T-cell depletion, C.I.; confidence interval
33
0.026
(1.19 – 15.30)
15/3
Group 3 In vivo TCD
0.026
< 0.001
Table 3
Risk factors for the development of secondary graft failure with complete
donor-type chimerism (A) All Patients Univariate analysis***
P value Donor type Group 0
146/13/6/59/7/24/47
/MUD88* /MUD66*/MUD*/MUD87
Group 4
2/0/1/6/1/1/4
CY dose low/middle/high**
Group 0
94/116/87
MRD/1MM-RD/23MM-RD
0.027
0.069
Group 4 Flu use
+/-
Group 0 Group 4
In vivo TCD +/-
Group 0 Group 4
5/10/1 175/143 0.016 13/2 177/158 0.079 13/4
* Matched unrelated donor 88 (MUD88) included donors with human leukocyte antigen (HLA) -A, -B, -C, -DRB1 8/8 allele-match, and MUD66 included donors with HLA -A, -B, -DRB1 6/6 allele-match. MUD included donors with HLA -A, -B, -DR 6/6 antigen-match, and MUD88, MUD66, or one allele-mismatched in HLA-A, -B, -C, -DRB1 unrelated donor (MUD87) were excluded. ** The dose of cyclophosphamide (CY) was classified as follows; low: less than 100 mg/kg, middle; 100 mg/kg or more than 100 mg/kg, but less than 200 mg/kg, high; 200 mg/kg or more than 200 mg/kg. *** Significant factor was not detected by a multivariate analysis. Abbreviations: MRD; matched related donor, 1MM-RD; HLA one antigen-mismatched in graft-versus-host direction related donor, 1MM-RD; HLA 2-3 antigen-mismatched in graft-versus-host direction related donor, Flu; fludarabine, TCD; T-cell depletion
34
(B) Patients who received cyclophosphamide in conditioning regimens
Univariate analysis P value
Multivariate analysis Odds ratio
P value
(95% C.I.) Donor type MRD/1MM-RD/23MM-RD /MUD88*/MUD66*/MUD*/MUD87
CY dose
Flu use
low/middle/high**
+/－
In vivo TCD
+/－
Group 0
140/13/2/54/7/23/45
Group 4
2/0/1/6/1/1/4
Group 0
78/116/87
Group 4
5/10/1
Group 0
157/142
Group 4
13/2
Group 0
172/144
Group 4
13/4
0.012
0.069
0.014
8.68 (1.11 - 67.80)
0.084
* Matched unrelated donor 88 (MUD88) included donors with human leukocyte antigen (HLA) -A, -B, -C, -DRB1 8/8 allele-match, and MUD66 included donors with HLA -A, -B, -DRB1 6/6 allele-match. MUD included donors with HLA -A, -B, -DR 6/6 antigen-match, and MUD88, MUD66, or one allele-mismatched in HLA-A, -B, -C, -DRB1 unrelated donor (MUD87) were excluded. ** The dose of cyclophosphamide (CY) was classified as follows; low: less than 100 mg/kg, middle; 100 mg/kg or more than 100 mg/kg, but less than 200 mg/kg, high; 200 mg/kg or more than 200 mg/kg. Abbreviations: MRD; matched related donor, 1MM-RD; HLA one antigen-mismatched in graft-versus-host direction related donor, 1MM-RD; HLA 2-3 antigen-mismatched in graft-versus-host direction related donor, Flu; fludarabine, TCD; T-cell depletion
35
0.039
Table 4 Outcomes of interventions for secondary graft failure
Group 3
Group 4
No. of Patients (effective)
No. of Patients (effective)
Observation
5 ( 2)
3 (2)
Increase in IS
3 (1)
7 (5)
Decrease in IS
8 (1)
3 (0)
DLI
2 (0)
1 (0)
12 (10)
8 (4)
Second transplantation
Abbreviations: IS; immunosuppressant, DLI; donor lymphocyte infusion
36