Peripheral blood stem cell mobilization and apheresis: analysis of adverse events in 94 normal donors

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1 Bone Marrow Transplantation, (1999) 24, Stockton Press All rights reserved /99 $ Peripheral blood stem cell mobilization and apheresis: analysis of adverse events in 94 normal donors M Murata 1,2, M Harada 3, S Kato 4, S Takahashi 5, H Ogawa 6, S Okamoto 7, S Tsuchiya 8, H Sakamaki 9, Y Akiyama 10 and Y Kodera 1 1 Division of Hematology, Department of Internal Medicine, Japanese Red Cross Nagoya First Hospital, Nagoya; 2 First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya; 3 Second Department of Internal Medicine, Okayama University Medical School, Okayama; 4 Department of Pediatrics, Tokai University School of Medicine, Kanagawa; 5 Internal Medicine, Institute of Medical Science, University of Tokyo, Tokyo; 6 Department of Medicine III, Osaka University Medical School, Osaka; 7 Division of Hematology, Keio University School of Medicine, Tokyo; 8 Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai; 9 The Bone Marrow Transplantation Team, Tokyo Metropolitan Komagome Hospital, Tokyo; and 10 Department of Pediatrics, Kyoto University, Kyoto, Japan Summary: Adverse events were analyzed in 94 normal donors who underwent PBSC harvest with G-CSF. The median dose of G-CSF was 9.7 g/kg/day (range, ), and the duration of administration was 4 6 days. Frequent symptoms were bone pain (71%), general fatigue (33%), headache (28%), insomnia (14%), anorexia (11%), nausea and/or vomiting (11%). One donor (1%) developed grade 3 toxicity bone pain (WHO criteria). WBC counts and ANC increased during G-CSF administration. After leukapheresis, three donors (3%) developed grade 3 toxicity neutropenia. Platelet counts decreased after leukapheresis. Three donors (3%) developed grade 3 thrombocytopenia. The means of both ALP and LDH increased approximately 1.9-fold compared with pretreatment levels. In one pediatric donor (1%), ALP was elevated to the grade 3 toxicity level. From multivariate analysis, the incidence of bone pain increased when G- CSF was given at a dose of 8.8 g/kg/day or more, headaches were frequent in donors younger than 35 years, and the incidence of nausea and/or vomiting was high in female donors. The peak levels of WBC counts and ANC and post-treatment level of LDH increased in correspondence with the escalation of G-CSF dose. All adverse events normalized on follow-up evaluation. In conclusion, although PBSC harvest for normal donors is acceptable, care must be taken for all donors in terms of their sex and age as well as the G-CSF dose. We recommend less than 8.8 g/kg/day as the G-CSF dose for PBSC mobilization in normal donors. Keywords: granulocyte colony-stimulating factor; peripheral blood stem cells; mobilization; apheresis; allogeneic transplantation; donor Correspondence: Dr M Murata, First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan Received 21 December 1998; accepted 30 June 1999 Attention has focused on allogeneic transplantation of peripheral blood stem cells (PBSC). 1 4 Recent studies have shown that it results in rapid hematological and immunological recovery without excessive acute graft-versus-host disease compared with bone marrow transplantation This contributes to less transfusion before hematological recovery, early discharge, and lower transplantation cost. 5 For the donor, the mobilization of PBSC and collection by leukapheresis has practical advantages over the traditional marrow harvest, including the avoidance of general anesthesia, absolute blood loss and invasive surgery. Adverse events in normal donors include bone pain, general fatigue, headache, nausea and changes in their own laboratory data (eg elevation in lactate dehydrogenase and alkaline phosphatase) with PBSC mobilization and apheresis However, the cumulative number of normal donors treated so far has been relatively small, and is still not sufficient to evaluate safety satisfactorily. Specific information regarding toxicity has also been limited. Furthermore, the risk factors for adverse events have not been clearly delineated. To determine the incidence of each adverse event and to analyze risk factors, we studied adverse events in 94 normal donors who donated their PBSC for allogeneic transplantation. From our analysis, the donor s sex and age as well as the dose of granulocyte colony-stimulating factor (G-CSF) correlated with certain adverse events. The results should be helpful in predicting the type of adverse event, in selecting only one donor when there are several candidates, and in determining the appropriate dose of G-CSF for PBSC mobilization. Materials and methods Study design This study was a retrospective analysis of 94 normal donors who underwent PBSC harvest between February 1992 and August All clinical records were reviewed to ascertain the donor s age, sex, weight, methods of mobilization and apheresis, and adverse events. Data were collected in March 1998, and informed consent was obtained from all donors.

2 1066 Donors The study population consisted of 52 males and 42 females (Table 1). The median age of donors was 36 years (range, 3 70), and the median weight was 58 kg (range, 21 90). Twenty-nine (31%) donors had experienced bone marrow harvest before PBSC harvest. Statistical analysis Adverse events were evaluated according to WHO criteria. In multivariate analysis for the incidence of each symptom, a logistic model was used with the variables of sex, age and dose of G-CSF. Selection of the factors with an important effect on the incidence of each symtom was based on a forward stepwise procedure. Complete blood counts and serum chemistry measures are presented as mean 1 s.d. The differences between pre- and post-treatment laboratory data were analyzed by a paired t-test. In assessing the association of the three variables with the post-treatment laboratory data, we used multiple linear regression analysis. For all analyses, a two-tailed P value 0.05 was considered significant. Results Mobilization and apheresis All donors were mobilized with subcutaneous administration of G-CSF. The dose and duration were in accord- Table 1 Characteristics of 94 normal donors. No. (%) Sex Male 52 (55) Female 42 (45) Age (years) 9 9 (10) (10) (15) (24) (28) (11) 60 2 (2) Body weight (kg) (13) (48) (34) 80 5 (5) Dose of G-CSF administered ( g/kg/day) (7) (48) (43) 15 2 (2) Duration of G-CSF administration 4 days 15 (16) 5 days 62 (66) 6 days 17 (18) First day of apheresis Day 4 66 (70) Day 5 25 (27) Day 6 3 (3) ance with each institution s standard regimen. Doses were calculated on the basis of donor s real weight ( g/kg) and summarized in Table 1. The median dose was 9.7 g/kg/day (range, ). Fifty-one (54%) donors received G-CSF twice a day, while 43 (46%) donors received it once a day. Physicians or nurses in each institution administered G-CSF to all donors. In 85 (90%) donors the peripheral vein was used for apheresis. Eight (9%) out of 85 donors needed the central vein as an access route. One (1%) donor underwent apheresis through a radial artery. All donors completed apheresis. The median volume of apheresis was 154 ml/kg/day (range, ). Symptoms Symptoms during G-CSF administration and apheresis procedure are summarized in Table 2. Bone pain was the most frequent symptom. One donor (1%) developed grade 3 toxicity bone pain, which resolved with an opiate. Other symptoms frequently experienced by more than 10% of donors were general fatigue, headache, insomnia, anorexia, nausea and/or vomiting. All symptoms were resolved after the apheresis procedure. Several variables were examined by multivariate analysis for their effect on the incidence of each symptom. Those found to be significant are presented in Table 3. When G-CSF was given at a dose of 8.8 g/kg/day or more, the incidence of bone pain increased. In donors younger than 35 years, the incidence of headaches was high. Women were more likely to experience nausea and/or vomiting. We also analyzed the effect of divided G-CSF administration (twice a day) and experience of marrow harvest before PBSC harvest on bone pain. There were no statistically significant differences between G-CSF administration once or twice a day, or between having or not having a marrow harvest before PBSC harvest. No changes in electrocardiograms (n 28) or chest X-ray photographs (n 9) were observed during or after the PBSC harvest procedure. White blood cells (WBC) counts and their subsets In all evaluable donors (n 84) G-CSF administration caused an elevation of WBC counts (Figure 1). On day 5 the median WBC count reached a maximum ( / l; P compared to pretreatment level), and on day 10 the median WBC count returned to the pretreatment level (pretreatment, / l; day 10, / l; P 0.59). We assessed the association of sex, age and G-CSF dose with the peak WBC counts. The peak WBC counts increased in correspondence with the escalation of G-CSF dose (coefficient, 3015; 95% confidence interval, ; P ). Neither sex nor age affected the peak WBC counts. Absolute neutrophil count (ANC) and other subsets Changes of ANC in 82 normal donors are shown in Figure 2. On day 5 the median ANC reached a maximum ( / l; P compared to pretreatment level). The peak level of ANC increased in correspondence with

3 Table 2 Symptoms in 94 normal donors with G-CSF administration and apheresis Grade (WHO) Total number (%) Bone pain (71) General fatigue (33) Headache (28) Insomnia (14) Anorexia (11) Nausea/vomiting (11) Paresthesia a (5) Diarrhea (3) Fever (2) Thirst (1) Constipation (1) a Paresthesias of the fingers, toes, and circumoral region. Table 3 Multiple logistic regression analysis of risk factor for symptoms Symptom Independent variable Relative risk P value (risk factor) 95% CI Bone pain G-CSF 8.8 g/kg/day Headache Age 35 years Nausea/vomiting Female WBC counts (109/l) Figure 1 1 s.d. pre Days WBC counts in 84 normal donors. Values represent mean ANC (109/l) Figure 2 pre Days ANC in 82 normal donors. Values represent mean 1 s.d. increased during G-CSF administration, while basophil counts did not change. the escalation of G-CSF dose (coefficient, 2536; 95% confidence interval, ; P ). Neither sex nor age affected the peak ANC. Although there was no statistically significant difference, the ANC on day 14 and 21 fell below the pretreatment levels (pretreatment, / l; day 14, / l; day 21, / l; P 0.15 and 0.84, respectively, compared to pretreatment level). In three donors (3%) the ANC on day 14 or 21 fell to less than 1000/ l (grade 3 toxicity). All neutropenia was asymptomatic and had resolved on follow-up evaluation. Eosinophil, monocyte and lymphocyte counts transiently Platelet counts The changes in platelet counts in 84 normal donors are shown in Figure 3. The platelet counts gradually decreased during G-CSF administration and dropped after leukapheresis. The comparison of platelet counts among 72 evaluable donors on the day before and after the first leukapheresis demonstrated a significant difference ( / l vs / l; P ; by paired t-test analysis). In three donors (3%) the platelet counts fell to less than / l (grade 3 toxicity), but no bleeding tendency was observed. The mean platelet counts on day

4 1068 Platelet counts (109/l) pre other donors developed grade 2 or less toxicity. We assessed the association of sex, age and dose of G-CSF with the post-treatment serum chemistry levels. Those found to be significant are presented in Table 5. The post-treatment level of LDH increased together with the escalation of G- CSF dose. As the donor s age increased, the post-treatment BUN level increased, whereas that of ALP decreased. The post-treatment levels of UA and CRP in men were higher than in women. Figure 3 1 s.d. Days Platelet counts in 84 normal donors. Values represent mean 21 were higher than the pretreatment level (pretreatment, / l; day 21, / l; P ), and normalized on follow-up evaluation. Hemoglobin concentration Hemoglobin concentration gradually decreased until day 14 and normalized on follow-up evaluation. The pretreatment and lowest hemoglobin levels were and g/dl, respectively, reflecting a significant difference (P ). Serum chemistries The effects of G-CSF administration on serum chemistries are summarized in Table 4. G-CSF caused increases in alkaline phosphatase (ALP), lactate dehydrogenase (LDH), uric acid (UA), sodium, and C-reactive protein (CRP). Decreases occurred in total protein, albumin, total-bilirubin (T-bil), blood urea nitrogen (BUN), potassium and chloride. The means of both ALP and LDH increased approximately 1.9-fold compared with pretreatment levels. In one pediatric donor (1%) ALP increased to grade 3 toxicity, while Follow-up evaluation We performed follow-up evaluations on 20 donors 6 12 months after PBSC harvest. None of the donors showed any symptoms associated with PBSC harvest. One donor, a 44-year-old man, developed gastric cancer 2 years after donation, but he had already complained of stomach discomfort before PBSC harvest. We measured WBC counts, ANC, platelet counts and hemoglobin concentrations in eight donors 6 12 months after PBSC harvest. The medians were, respectively, 6900 (range, )/ l, 2900 ( )/ l, 24.2 ( ) 10 4 / l, and 13.7 ( )g/dl. Discussion The symptoms consisted mainly of bone pain, general fatigue, headaches, insomnia, anorexia, and nausea and/or vomiting. Most of them were grade 2 or less toxicity, and all symptoms resolved after the apheresis procedure. The symptoms associated with PBSC harvest were acceptable for normal donors. Bone pain was the most common symptom experienced by the 94 donors. Some reports have demonstrated a relationship between G-CSF dose and the frequency of bone pain. 16,19 In our study the incidence of bone pain was Table 4 Changes in serum chemistries Pretreatment level Post-treatment level P value No. a Total protein (g/dl) Albumin (g/dl) AST (IU/l) ALT (IU/l) ALP (IU/l) LDH (IU/dl) T-bil (mg/dl) BUN (mg/dl) Creatinine (mg/dl) UA (mg/dl) Sodium (meq/l) Potassium (meq/l) Chloride (meq/l) Calcium (mg/dl) Phosphorus (mg/dl) CRP (mg/dl) a Number of donors tested. AST aspartate aminotransferase; ALT alanine aminotransferase. Other abbreviations are presented in the text. Values given are mean 1 s.d.

5 Table 5 Multiple linear regression analysis of risk factors for changes in serum chemistries Independent variable Coefficient 95% confidence interval P value 1069 ALP Age LDH Dose of G-CSF BUN Age UA Male CRP Male high in normal donors given G-CSF at a dose of 8.8 g/kg/day or more. Divided administration of G-CSF (twice a day) was not effective for decreasing bone pain. Although bone pain can usually be managed successfully with analgesics, it is desirable to give G-CSF at a dose of less than 8.8 g/kg/day. Interestingly, there was no difference in the incidence of bone pain between having or not having a marrow harvest before PBSC harvest. Through the National Marrow Donor Program (NMDP), in some cases when marrow graft failure occurred, some unrelated marrow donors have been given G-CSF and their PBSC have been collected for a second transplant. The second donation after bone marrow harvest would not be a risk factor for bone pain with G-CSF mobilization. No investigators have reported any association between the donor s age and headaches. The incidence of headaches was high in donors younger than 35 years. Hyperleukocytosis leading to hyperviscosity is known to cause headaches during G-CSF administration. 20 In our study population the peak WBC count in donors younger than 35 years was slightly less than in other donors ( and / l, respectively). There could be some etiology other than hyperleukocytosis. We also observed that women were more likely to experience nausea and/or vomiting, in agreement with the previous report. 16 Due care must be taken for donors in terms of their sex and age as well as the G-CSF dose. The means of both ALP and LDH increased approximately 1.9-fold compared with pretreatment levels. These changes are consistent with the expansion of the marrow pool of myeloid progenitors. We found correlations between some kinds of serum chemistries and sex, age and dose of G-CSF (Table 5). It is known that the standard ALP level is high in young people, especially in children, that BUN is high in the elderly, and that UA is high in men. The increase in LDH is probably caused by the expansion of myeloid progenitors with a higher dose of G-CSF. The CRP level increases in conditions characterized by inflammation with tissue destruction. It is possible that men are more susceptible to inflammation caused by G-CSF than women. None of these changes indicate that the use of G-CSF for PBSC mobilization causes undue risk to the donors. We found a transient decrease in ANC after the whole PBSC harvest procedure. However, no neutropenia reported, including that in our results, was associated with symptoms or infectious complications, and resolved on follow-up evaluation. 15,21 The major cause of neutropenia is thought to be a negative feedback effect after marked neutrophilic leukocytosis, but the precise mechanisms remain unclear. Although there was no clinical evidence of bleeding in any of the 94 donors, the platelet counts fell below pretreatment levels after apheresis and remained lower until day 10. Thrombocytopenia is sometimes prolonged for a few days after leukapheresis. 22 After one apheresis, the platelet counts decreased by 33%, from to / l. If the percentage of platelets lost is similar for each collection, our results suggest that platelet counts will fall to about / l after the second apheresis. The decrease in platelet counts could limit the number of aphereses. We recommend that the platelet count should be done before each apheresis, and the next collection should be cancelled if less than / l (grade 2). The long-term effects of G-CSF in normal donors are presently unknown. 13,23 In the present study, one donor developed gastric cancer 2 years after PBSC harvest. Because this donor had already complained of stomach discomfort before PBSC harvest, the cancer had probably developed before G-CSF administration. Receptors for G- CSF have not been found on the surface of adenocarcinoma cells of the stomach, and its administration combined with high-dose chemotherapy does not induce the growth of cancer. 24 One of the major advantages of allogeneic peripheral blood stem cell transplantation over marrow transplantation is that for the donor the risks associated with general anesthesia and certain amount of blood loss, and discomfort caused by multiple bone punctures are avoided. According to the report from the NMDP, adverse events experienced by 433 donors 7 14 days after marrow harvest included pain at the donation site (87%), fatigue (80%), low back pain (73%), and difficulty in walking (66%). 25 The data reported here confirm the expectation that the short-term and medium-term (1 year) effects of PBSC harvest were acceptable for normal donors. However, several other adverse effects of G-CSF administration, which were not observed in our study, have been reported. Its administration has activated symptoms of autoimmune disorders, 26 mobilized osteoclast progenitors in the peripheral blood, 27 and caused splenic rupture. 28 Furthermore, the maximum serum concentration of G-CSF for mobilization is far higher than that of endogenous G-CSF in human subjects under physiologic or pathophysiologic conditions. 13 All donors to be given G-CSF should be informed of its potential adverse effects. It is important to determine the appropriate dose of G- CSF for mobilization in normal donors. From our multivariate analysis, the dose affects the incidence of bone pain,

6 1070 the peak WBC counts and ANC, and the post-treatment level of LDH. The reason for G-CSF use is to increase WBC counts and ANC, but at the same time the excessive results inevitably mean adverse events as well. The transient 1.9-fold increase in LDH is of no clinical significance. Bone pain is a major factor in determining the G-CSF dose. If a sufficient number of PBSC can be obtained for reliable, rapid and sustained engraftment in allogeneic transplantation of PBSC (something as yet to be determined), G-CSF at a dose of less than 8.8 g/kg/day should be recommended. In conclusion, although the short- and medium-term effects of PBSC harvest are acceptable, due care must be taken for all normal donors in terms of their sex and age as well as G-CSF dose, and they should be monitored closely over the long term. Acknowledgements This work was supported by the Grant-in-aid for Cancer Research (7 3) from the Ministry of Health and Welfare, Japan. We are grateful to Dr K Wakai for his statistical analysis and to Dr R Suzuki for his helpful discussion. References 1 Azevedo WM, Aranha FJP, Gouvea JV et al. Allogeneic transplantation with blood stem cells mobilized by rhg-csf for hematological malignancies. Bone Marrow Transplant 1995; 16: Bensinger WI, Weaver CH, Appelbaum FR et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1995; 85: Korbling M, Przepiorka D, Huh YO et al. Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 1995; 85: Schmitz N, Dreger P, Suttorp M et al. Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor). Blood 1995; 85: Bensinger WI, Clift R, Martin P et al. 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Allogeneic bone marrow transplantation vs filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicentre trial of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 1998; 21: Ottinger HD, Beelen DW, Scheulen B et al. Improved immune reconstitution after allotransplantation of peripheral blood stem cells instead of bone marrow. Blood 1996; 88: Waller CF, Bertz H, Wenger MK et al. Mobilization of peripheral blood progenitor cells for allogeneic transplantation: efficacy and toxicity of a high-dose rhg-csf regimen. Bone Marrow Transplant 1996; 18: Anderlini P, Przepiorka D, Champlin R, Korbling M. Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 1996; 88: Anderlini P, Przepiorka D, Seong D et al. 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7 28 Becker PS, Wagle M, Matous S et al. Spontaneous splenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): occurrence in an allogeneic donor of peripheral blood stem cells. Biol Blood Marrow Transplant 1997; 3: Appendix Institutions participating in this study Division of Hematology, Department of Internal Medicine (Y Kodera and M Murata), and Division of Hematology and Oncology, Children s Medical Center (T Matsuyama), Japanese Red Cross Nagoya First Hospital, Nagoya, Japan; Second Department of Internal Medicine, Okayama University Medical School, Okayama, Japan (M Harada); Department of Pediatrics (S Kato), and Fourth Department of Internal Medicine (T Hotta), Tokai University School of Medicine, Kanagawa, Japan; Internal Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan (S Takahashi); Department of Medicine III, Osaka University Medical School, Osaka, Japan (H Ogawa); Division of Hematology, Keio University School of Medicine, Tokyo, Japan (S Okamoto); Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan (S Tsuchiya); The Bone Marrow Transplantation Team, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan (H Sakamaki); Department of Pediatrics, Kyoto University, Kyoto, Japan (Y Akiyama); Department of Pediatrics, Ibaragi Children s Hospital, Ibaragi, Japan (M Tsuchida); Department of Hematology and Chemotherapy, Kanagawa Cancer Center, Kanagawa, Japan (A Maruta); Bone Marrow Transplantation Division, Niigata University Medical Hospital, Niigata, Japan (T Furukawa); Hematology and Bone Marrow Transplantation Center, Meitetsu Hospital, Nagoya, Japan (H Sao); Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan (N Imamura); Department of Medical Oncology, National Cancer Center, Tokyo, Japan (Y Kobayashi); Department of Hematology/Oncology, Jikei University Hospital, Tokyo, Japan (O Asai); Third Department of Internal Medicine, Kinki University School of Medicine, Osaka, Japan (A Kanamaru); Division of Hematology and Oncology, Hyogo Medical Center for Adults, Hyogo, Japan (T Nakagawa).