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Directed sibling donor cord blood banking for children with β-thalassemia major in Greece: Usage rate and outcome of transplantation for HLA-matched units
Blood Cells, Molecules, and Diseases 44 (2010) 107–110
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
Directed sibling donor cord blood banking for children with β-thalassemia major in Greece: Usage rate and outcome of transplantation for HLA-matched units Evgenios Goussetis a,⁎, Eftichia Petrakou a, Maria Theodosaki a, Vasiliki Kitra a, Ioulia Peristeri a, George Vessalas a, Maria N. Dimopoulou a, Antonia Spiropoulos a, Andreas C. Papassavas b, Catherine Stavropoulos-Giokas b, Stelios Graphakos a a b
Stem Cell Transplant Unit, Saint Sofia Children's Hospital, Thivon and Mikras Asias, 11527 Athens, Greece Hellenic Cord Blood Bank, Foundation for Biomedical Research, Academy of Athens, Athens, Greece
a r t i c l e
i n f o
Article history: Submitted 3 October 2009
(Communicated by G. Stamatoyannopoulos, M.D., Dr. Sci., 21 October 2009) Keywords: Cord blood Banking Transplantation β-thalassemia
a b s t r a c t Several cord blood banks store cord blood units from healthy siblings of patients, who are candidates for stem cell transplantation. We analyzed the quality characteristics of 50 cord blood units collected from families with β-thalassemia major and the outcome of subsequent stem cell transplantations during a 15year period. All cord blood units were found suitable for banking based on a minimum net volume of 40 ml. The mean volume of the units was 98.9 ml; the mean total nucleated cell count (NC) was 7.8 × 108 and the mean CD34+ cell count was 2.8 × 106. Eight out of twelve HLA matched collections were released for transplantation. All but one recipient belonged to Pesaro II-III risk classes. Three patients received a cord blood graft with N 5 × 107 NC/kg . One of them with Pesaro class I disease engrafted, whereas the other two who failed to engraft, were re-transplanted with bone marrow from the same donor later. Cord blood grafts containing NCs b 4 × 107/kg combined with reduced volume bone marrow from the same donor were used in all 5 remaining cases and stable engraftment was achieved. All patients survived, 7/8 thalassemia-free. Cord blood banking from healthy siblings of children with β-thalassemia major can result in a successful transplantation in cases in which there is HLA compatibility. However, in high-risk patients, the use of combined cord blood and bone marrow grafts seems necessary in order to ensure stable engraftment, especially when cord blood unit cell counts are low. © 2009 Elsevier Inc. All rights reserved.
Introduction Bone marrow transplantation from an HLA-matched sibling has achieved long-term cure of β-thalassemia major for 80–90% of patients, if performed early in life [1–3]. However, the majority of young βthalassemia patients are not eligible for related bone marrow transplantation mainly because they do not have an HLA-identical sibling donor. For such patients, mismatched family members and matched unrelated donors can be considered as alternative donors. Data obtained from small numbers of patients showed that the outcome of these transplants has been confounded by an increase of transplantrelated complications including early and late toxicity, mortality, and rejection [4,5]. When families with thalassemia conceive a healthy fully matched sibling, cord blood banking and subsequent cord blood transplantation could spare healthy sibling donors from the morbidity of bone marrow donation. The experience on cord blood transplantation for thalassemia, using HLA-matched siblings is quite limited. However, the results of the small series published to date are encouraging as they appear to be comparable to the ones achieved with bone marrow transplantation [6,7]. The largest series comes from a multicentre study ⁎ Corresponding author. Fax: +30 210 7792200. E-mail address: mmo@paidon-agiasofia.gr (E. Goussetis). 1079-9796/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2009.10.011
on behalf of the Eurocord group, which reported an overall survival 100% and a 2-year estimate of event-free survival 79% [6]. The main disadvantage of cord blood was the high risk of graft failure, whereas the low treatment-related mortality and short-term morbidity were major advantages of cord blood transplantation for patients with thalassemia major. More data are necessary to draw firm conclusions about the exact role of cord blood transplantation for the treatment of thalassemia. Currently, several cord blood banks offer cord blood unit storage to families who already have a child with a disease that is potentially curable with cord blood transplantation and are expecting another baby [8–10]. We report our experience on directed sibling cord blood banking for patients with β-thalassemia major, the likelihood of using a matched cord blood collection either alone or in combination with bone marrow from the same donor, and the outcome of such transplants in Greece. Patients and methods Eligibility for directed sibling cord blood banking, written consent and cord blood collection Families who already had a child with β-thalassemia major candidate for stem cell transplantation and expecting the birth of a healthy
108
E. Goussetis et al. / Blood Cells, Molecules, and Diseases 44 (2010) 107–110
Table 1 Laboratory characteristics of banked sibling cord blood units.
Volume in ml (w/35 ml anticoagulant) Nucleated cell count × 108 Total CD34+ cells/collection × 106 (n = 28) Viability %
Mean
SD
Range
98.9 7.8 2.8 98.6
31.1 5.1 1.9 1.6
(40–150) (2–20) (0.8–8.4) (88–100)
sibling were eligible for cord blood collection. Families were enrolled in an approved research protocol; written informed consent was obtained from the donor mother for screening of both the mother and the cord blood for mandatory microbiological markers, for tissue typing, and for the storage of the cord blood units. Families were not charged for cord blood banking. Enrollment began May 01, 1994. Characteristics of the banked cord blood units are shown in Table 1. The cord blood was collected before delivery of the placenta using a 250-ml blood bag with 35 ml citrate-phosphate-dextrose anticoagulant. Cord blood units were considered suitable for banking based on a net volume of ≥40 ml since even for low volume collections there is the possibility of combining the cord blood unit with a small volume of bone marrow from the same donor. Blood samples for HLA-typing, blood grouping, testing for infectious and genetic diseases, and for total and CD34+ cells counting were removed. All cord blood units collected between 1994 and 2003 (n = 38) were processed without volume reduction and stored in the bone marrow processing laboratory of the Stem Cell Transplantation Unit at Saint Sofia Children's Hospital. Cryoprotectant solution was prepared from dimethyl sulfoxide (DMSO) and 5% (wt/vol) human albumin solution. An equal volume of cooled 20% (vol/vol) DMSO solution was added to the cord blood. Cord blood cells were cryopreserved in freezing bags (Gabro, Haemofreeze, Amsterdam) using an automated, controlled cell freezer (Cryo10, CryoMed). In 2004, governmental funding facilitated the establishment of the first national cord blood bank, where 12 additional cord blood units were processed and stored as described by Rubinstein et al. [11]. Briefly, cryoprotectant solution was prepared from dimethyl sulfoxide and 10% (wt/vol) dextran-40/ saline solution. An equal volume of sterile, cooled 20% (vol/vol) DMSO solution was added to the cord blood in a controlled manner. The cord blood units after completion of the freezing program were stored in the liquid phase of a liquid nitrogen freezer at − 196 °C. Aliquots were taken post-processing for blood culture testing (1.0 ml for aerobic and anaerobic culture) with an incubation period of 7 days. Patients, grafts and conditioning regimens Our patients were classified in 1 of 3 classes according to the Pesaro criteria proposed by Lucarelli et al. [12]. Risk factors were hepatomegaly, portal fibrosis in the liver biopsy, and quality of
chelation therapy. Eight children with β-thalassemia major, 1 class I, 2 class II, and 5 class III, were transplanted by infusing either cord blood cells alone (n = 3) or cord blood cells combined with bone marrow cells (n = 5) from their HLA-matched siblings. Bone marrow cells were used because the number of cord blood NCs was considered insufficient to ensure engraftment according to current recommendations on cord blood transplantation for using a minimum NC dose of 4 × 107/kg for hemoglobinopathies [13]. Thus, following parental consent, we harvested a reduced bone marrow volume (median value 250 ml, range 190–280 ml) from the siblings aged between 2 and 3 years old. In our centre, for the transplantation of a thalassemic patient with a marrow graft, a minimum dose of 4 × 108 NCs/kg is required. The estimated bone marrow volume for this cell dose is approximately 500 ml for older patients with body weight N30 kg. Because cord blood was already available from the donors we proceeded with collecting less than half the volume required in case the transplant was to be performed solely with bone marrow graft, corresponding to a mean dose of 1.8 × 108 NCs/kg. Six cord blood units were thawed in a 37 °C waterbath with gentle agitation and immediately infused into the patients without further processing. In 2 cases, due to a donor-recipient major ABO incompatibility, cord blood cells were separated into a low-density fraction using Ficoll– Hipaque (d = 1.077 g/ml) after thawing. For both these cases the decision of combining cord blood with bone marrow was already based on the initial cell counts and therefore we were not concerned about further cell losses because of the Ficoll separation. Patients, donors, and graft characteristics are shown in Table 2. Seven patients underwent transplantation after a preparative regimen including busulfan (16 mg/kg), cyclophosphamide (200 mg/kg) and antilymphocyte globulin pre-transplant (40 mg/kg). The remaining patient was given a conditioning regimen including busulfan, cyclophosphamide, anti-thymocyte globulin, and fludarabine. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine A from day −1 to 9 months and then tapered off over the subsequent 3 months. Myeloid engraftment was defined as the first of 3 consecutive days when the absolute neutrophil count was 0.5 × 109/ l or higher, and platelet engraftment as the time to reach a sustained platelet count of 20 × 109/l or higher in the absence of platelet transfusion for 7 consecutive days. Donor hematopoiesis was documented by PCR study of genetic polymorphism of short tandem repeats. Results All 50 cord blood units collected were banked. All banked cord blood units were free of bacterial contamination. HLA-A, HLA-B, and HLA-DR typing was performed serologically. DNA analysis was used only in one case because there was one mismatched antigen. Eleven cord blood units were HLA identical to the prospective recipient and
Table 2 Patients, grafts and transplant outcomes. Patient 1 2 3 4b 5b 6b 7 8
Age 3 6 3 5 14 15 15 7
Weight 13 20 13 16 62 50 56 20
Pesaro Class I III II II III III III III
HLA
CB-NCa
CB-CD34a
BM-NC
Outcome
id 1Ag mm id id id id id id
(×107/kg) 6.8 8 5.2 0.3c 0.4c 1.8 1.4 3.2
(×104/kg) ND ND 20 6 17 5 8 16
(×108/kg) – – – 0.7 1.6 1.7 1.2 3.8
ATF 14 y AR AR-TF ATF 13 y ATF 12 y ATF 11 y ATF 4 y ATF 3 y
CB: cord blood; BM: bone marrow; NC: nucleated cells; ND: not done; id: identical; 1Ag mm: 1 antigen mismatch; ATF: alive and thalassemia free; AR: alive with thalassemia after rejection; AR-TF: alive, thalassemia free after second transplant with BM; y: years post-transplant. a The cord blood nucleated cell (CB-NC) counts reported were performed post-thawing of the units. b Data from these recipients are previously published [19]. c The counts reported were performed after Ficoll separation.
E. Goussetis et al. / Blood Cells, Molecules, and Diseases 44 (2010) 107–110
one cord blood unit was mismatched for one HLA-A antigen. Out of 12 cord blood units with an acceptable HLA-match, 8 have been transplanted, one unit with a NC dose of 2 × 107/kg was not infused because a sufficient bone marrow graft (10 × 106CD34+ cells/kg) was given; and, three remaining cord blood units are planned to be given within the next 3 years. In three patients, class I, II, and III respectively according to the Pesaro classification, cord blood containing N5 × 107 NCs/kg was the sole stem cell source while in 5 patients (Pesaro class II or III) the infused grafts consisted of cord blood (after thawing and processing) and bone marrow, containing 1.4 ± 1.2 × 107 and 1.8 ± 1.1 × 108 NCs/kg, respectively. Among the 3 patients who received a cord blood allograft alone, two had HLA-identical donors and one had a donor mismatched at 1 HLA-A antigen. Only one child with Pesaro risk class I had a stable engraftment; the other two (including the patient with the 1 antigen mismatched graft) experienced primary graft failure. Both children, who failed to engraft, were re-transplanted with bone marrow at a later date from the same donor; one has been cured and the other one did not engraft and lives with thalassemia. In all 5 recipients of combined cord blood and bone marrow allografts, neutrophil engraftment occurred at a median of 18 days (range, 17–29) and platelet engraftment at a median of 24 days (range, 19–32) post-transplantation. Outcomes after transplantation are shown in Table 2. Discussion Since the beginning of our stem cell transplantation program for thalassemia patients in 1994, families with thalassemic children, who desire a new pregnancy, have been offered the possibility of collecting and subsequently using cord blood cells in transplantation. We enrolled 50 families in a national directed sibling cord blood banking program, and banked all cord blood units collected. Both volume and cell counts (nucleated cell and CD34+) of cord blood units are comparable to those reported by banks in United States and the United Kingdom involved in collection of cord blood units from siblings of patients with thalassemia [8,9]. Regarding the usage rate of cord blood units in stem cell transplantation, our results are also in line with the experience in those countries, confirming the very high utility rate of HLA-matched cord blood units. Walters et al. [14] described the experience in the United States, where 32/96 of the donor-recipient pairs with β-thalassemia (33%) were HLA-identical and 14 of them (44%) received cord blood transplantation either alone or in combination with bone marrow or blood progenitor cells (n = 4) from the same donor. Twelve out of fourteen patients survive diseasefree after transplantation. One patient died due to pulmonary toxicity and the other one had disease recurrence. Smythe et al. [9] have reported their 10-year experience with directed sibling cord blood banking in the National Blood Service in England and Wales. They have used 7 cord blood units out of 36 collected from families affected by β-thalassemia. All of their patients survived disease-free (one had graft rejection but engrafted after bone marrow transplantation from the same donor). Most cord blood units stored, however, share only one HLA-haplotype with the recipient and are unlikely to be used. Our policy is to advise the family to donate the mismatched cord blood unit to the unrelated cord blood units pool; otherwise we request that the unit is transferred to a private bank. Prenatal HLA typing would be a method of selecting pregnancies appropriate for banking [15]. Graft failure is a well documented complication after cord blood transplantation for β-thalassemia and the main risk factors are low cell dose, use of methotrexate, and inadequate immune suppression. It seems likely, from the results of stem cell transplantation for βthalassemia, that the high graft failure rate can be overcome by employing additional pretransplant immune suppression including fludarabin, anti-thymocyte globulin, and thiotepa. One recent study has reported no graft failure or rejection in 27 children with βthalassemia transplanted with a conditioning regimen consisting of
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busulfan, thiotepa and fludarabin without methotrexate for graftversus-host-disease prophylaxis [16]. In addition, results of a multicenter comparative study, published as an abstract, demonstrated a 5-year disease-free survival of 86 ± 5% after matched sibling cord blood transplantation in 48 patients with beta-thalassemia major vs. 91% ± 3% in 184 patients who received matching sibling bone marrow during the same period [17]. These data from both studies suggest that cord blood transplantation result in a similar outcome to bone marrow transplantation, the majority of patients transplanted with cord blood in all published studies were young children with goodrisk disease (Pesaro class 1). For high-risk patients (Pesaro class II and III) of older age, such as those in our study, the use of additional bone marrow stem cells has proven valuable, especially when the cellular content of the cord blood unit is judged insufficient to secure engraftment. In such cases, one may argue that it is quite possible to delay the transplant until the donor is old enough to donate sufficient number of bone marrow stem cells, thus making cord blood storage unnecessary. However, for older thalassemic recipients with high body weight, the advantage of combining cord blood with bone marrow cells is the reduction of the volume of bone marrow harvest required and avoiding a long delay for the transplantation of children with Pesaro risk class II or III who already have poor prognosis [18]. Reduced volume bone marrow collection not exceeding 15 ml/kg can be an attractive option for enhancing the stem cell number in the graft without placing the donor at the same risk as a standard bone marrow collection. Such an approach was effective and beneficial in our patients, three of whom weighed 62, 50, and 56 kg, respectively. Moreover, as we noted in a previous report, the evidence of possible hematopoietic reconstitution derived from cord blood cells, shown by elevated fetal hemoglobin (HbF) levels, could be the result of the cooperation between a small bone marrow inoculum and the cord blood cells [19]. Our results are in agreement with the recommendation of delaying cord blood transplantation until the HLA-identical donor is at least 2 years of age [20]. The use of combined cord blood and marrow cell grafts for Pesaro class II and III patients, who are in high risk of rejection, is reasonable where a transplantation using cord blood units with high cell counts (NCs N4 × 107/kg) as the only source of stem cells should be reserved for young children classified in the good risk group (Pesaro class I). The use of combined grafts has been reported previously, but the exact criteria for supplementing cord blood with bone marrow have not been established [21,22]. Despite the encouraging results reported to date, cord blood transplantation for thalassemia has several limitations. The usage rates of units collected in directed banking programs reported by us and others are within the range of 15–20%. In our hands the efficacy of using cord blood alone in high risk patients was not as high as previously reported with good risk patients. However we do believe that banking programs should be encouraged but efforts for acquiring better cell yields from cord blood unit collections need to be continued and strategies of facilitating engraftment need to be explored further. References [1] G. Lucarelli, M. Galimberti, C. Giardini, P. Polchi, E. Angelucci, D. Baronciani, et al., Bone marrow transplantation in thalassemia. The experience of Pesaro, Ann. N.Y. Acad. Sci. 850 (1998) 270–275. [2] S.E. Lawson, I.A. Roberts, P. Amrolia, I. Dokal, R. Szydlo, P.J. Darbyshire, Bone marrow transplantation for beta-thalassemia major: the UK experience in two paediatric centres, Br. J. Haematol. 120 (2003) 289–295. [3] E. Angelucci, D. Baronciani, Allogeneic stem cell transplantation for thalassemia major, Haematologica 93 (2008) 1780–1784. [4] G. La Nasa, F. Argiolu, C. Giardini, A. Pession, F. Fagioli, G. Caocci, et al., Unrelated bone marrow transplantation for beta-thalassemia patients: the experience of the Italian Bone Marrow Transplant Group, Ann. N.Y. Acad. Sci. 1054 (2005) 186–195. [5] D. Gaziev, M. Galimberti, G. Lucarelli, P. Polchi, C. Giardini, E. Angelucci, et al., Bone marrow transplantation from alternative donors for thalassemia: HLA-
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E. Goussetis et al. / Blood Cells, Molecules, and Diseases 44 (2010) 107–110 phenotypically identical relative and HLA-non-identical sibling or parent transplants, Bone Marrow Transplant. 25 (2000) 815–821. F. Locatelli, V. Rocha, W. Reed, F. Bernaudin, M. Ertem, S. Grafakos, et al., Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease, Blood 101 (2003) 2137–2143. J. Fang, S. Huang, C. Chen, D. Zhou, C.K. Li, Y. Li, et al., Umbilical cord blood transplantation in Chinese children with beta-thalassemia, J. Pediatr. Hematol. Oncol. 26 (2004) 185–189. W. Reed, R. Smith, F. Dekovic, J.Y. Lee, J.D. Saba, E. Trachtenberg, et al., Comprehensive banking of sibling donor cord blood for children with malignant and nonmalignant disease, Blood 101 (2003) 351–357. J. Smythe, S. Armitage, D. McDonald, D. Pamphilon, M. Guttridge, J. Brown, et al., Directed sibling cord blood banking for transplantation: the 10-year experience in the national blood service in England, Stem Cells 25 (2007) 2087–2093. T. Brune, H.S.P. Garritsen, Potential of directed cord blood donations for the treatment of patients with hematologic disorders, Transfus. Med. Hemother. 34 (2007) 100–103. P. Rubinstein, L. Dobrila, R.E. Rosenfield, J.W. Adamson, G. Migliaccio, A.R. Migliaccio, et al., Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 10119–10122. G. Lucarelli, A. Galimberti, P. Polchi, E. Angelucci, D. Baronciani, C. Giardini, et al., Bone marrow transplantation in patients with thalassemia, N. Engl. J. Med. 322 (1990) 417–421. E. Gluckman, V. Rocha, A. Boyer-Chammard, F. Locatelli, W. Arcese, R. Pasquini, et al., Eurocord Transplant and the European Blood and Marrow Transplantation Group, Outcome of cord-blood transplantation from related and unrelated donors, N. Engl. J. Med. 337 (1997) 373–381.
[14] M.C. Walters, L. Quirolo, E.T. Trachtenberger, S. Edwards, L. Hale, J. Lee, et al., Sibling donor cord blood transplantation for thalassemia major: experience of the sibling cord blood program, Ann. N.Y. Acad. Sci. 1054 (2005) 206–213. [15] H. van den Berg, M. Verjaal, K. Boer, N.M. Lardy, Prenatal HLA-matching to determine suitability for allogeneic bone marrow transplantation, Bone Marrow Transplant. 29 (2000) 527–529. [16] D. Lisini, M. Zecca, G. Giorgiani, D. Montagna, R. Cristantiell, M. Labirio, et al., Donor/recipient mixed chimerism does not predict graft failure in children with β-thalassemia given an allogeneic cord blood transplant from an HLA-identical sibling, Haematologica 93 (2008) 1859–1867. [17] N. Kabbara, F. Locatelli, V. Rocha, A. Ghavamzadeh, F. Bernaudin, C.K. Li, et al., A multicentric comparative analysis of outcome of HLA identical related cord blood and bone marrow transplantation with beta-thalassemia or sickle cell disease [Abstract], Biol. Blood Marrow Transplant. 14 (Suppl.2) (2008) 3–4. [18] G. Lucarelli, M. Andreani, E. Angelucci, The cure of thalassemia by bone marrow transplantation, Blood Rev. 16 (2002) 81–85. [19] E. Goussetis, J. Peristeri, V. Kitra, A. Kattamis, D. Petropoulos, I. Papassotiriou, et al., Combined umbilical cord blood and bone marrow transplantation in the treatment of β-thalassemia major, Pediatr. Hematol. Oncol. 17 (2000) 307–314. [20] F.O. Pinto, I. Roberts, Cord blood stem cell transplantation for hemoglobinopathies, Br. J. Haematol. 141 (2008) 309–324. [21] S. Dallorso, C. Dufour, F. Bertolini, G. Dini, G. Sirchia, P.G. Mori, Combined transplantation with related HLA-identical cord blood and bone marrow in a child with severe acquired aplastic anemia, Eur. J. Hematol. 56 (1996) 256–258. [22] F. Bernaudin, G. Socie, M. Kuentz, S. Chevret, M. Duval, Y. Bertrand, et al., Longterm results of related, myeloablative stem cell transplantation to cure sickle cell disease, Blood 110 (2007) 2749–2756.
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
Directed sibling donor cord blood banking for children with β-thalassemia major in Greece: Usage rate and outcome of transplantation for HLA-matched units Evgenios Goussetis a,⁎, Eftichia Petrakou a, Maria Theodosaki a, Vasiliki Kitra a, Ioulia Peristeri a, George Vessalas a, Maria N. Dimopoulou a, Antonia Spiropoulos a, Andreas C. Papassavas b, Catherine Stavropoulos-Giokas b, Stelios Graphakos a a b
Stem Cell Transplant Unit, Saint Sofia Children's Hospital, Thivon and Mikras Asias, 11527 Athens, Greece Hellenic Cord Blood Bank, Foundation for Biomedical Research, Academy of Athens, Athens, Greece
a r t i c l e
i n f o
Article history: Submitted 3 October 2009
(Communicated by G. Stamatoyannopoulos, M.D., Dr. Sci., 21 October 2009) Keywords: Cord blood Banking Transplantation β-thalassemia
a b s t r a c t Several cord blood banks store cord blood units from healthy siblings of patients, who are candidates for stem cell transplantation. We analyzed the quality characteristics of 50 cord blood units collected from families with β-thalassemia major and the outcome of subsequent stem cell transplantations during a 15year period. All cord blood units were found suitable for banking based on a minimum net volume of 40 ml. The mean volume of the units was 98.9 ml; the mean total nucleated cell count (NC) was 7.8 × 108 and the mean CD34+ cell count was 2.8 × 106. Eight out of twelve HLA matched collections were released for transplantation. All but one recipient belonged to Pesaro II-III risk classes. Three patients received a cord blood graft with N 5 × 107 NC/kg . One of them with Pesaro class I disease engrafted, whereas the other two who failed to engraft, were re-transplanted with bone marrow from the same donor later. Cord blood grafts containing NCs b 4 × 107/kg combined with reduced volume bone marrow from the same donor were used in all 5 remaining cases and stable engraftment was achieved. All patients survived, 7/8 thalassemia-free. Cord blood banking from healthy siblings of children with β-thalassemia major can result in a successful transplantation in cases in which there is HLA compatibility. However, in high-risk patients, the use of combined cord blood and bone marrow grafts seems necessary in order to ensure stable engraftment, especially when cord blood unit cell counts are low. © 2009 Elsevier Inc. All rights reserved.
Introduction Bone marrow transplantation from an HLA-matched sibling has achieved long-term cure of β-thalassemia major for 80–90% of patients, if performed early in life [1–3]. However, the majority of young βthalassemia patients are not eligible for related bone marrow transplantation mainly because they do not have an HLA-identical sibling donor. For such patients, mismatched family members and matched unrelated donors can be considered as alternative donors. Data obtained from small numbers of patients showed that the outcome of these transplants has been confounded by an increase of transplantrelated complications including early and late toxicity, mortality, and rejection [4,5]. When families with thalassemia conceive a healthy fully matched sibling, cord blood banking and subsequent cord blood transplantation could spare healthy sibling donors from the morbidity of bone marrow donation. The experience on cord blood transplantation for thalassemia, using HLA-matched siblings is quite limited. However, the results of the small series published to date are encouraging as they appear to be comparable to the ones achieved with bone marrow transplantation [6,7]. The largest series comes from a multicentre study ⁎ Corresponding author. Fax: +30 210 7792200. E-mail address: mmo@paidon-agiasofia.gr (E. Goussetis). 1079-9796/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2009.10.011
on behalf of the Eurocord group, which reported an overall survival 100% and a 2-year estimate of event-free survival 79% [6]. The main disadvantage of cord blood was the high risk of graft failure, whereas the low treatment-related mortality and short-term morbidity were major advantages of cord blood transplantation for patients with thalassemia major. More data are necessary to draw firm conclusions about the exact role of cord blood transplantation for the treatment of thalassemia. Currently, several cord blood banks offer cord blood unit storage to families who already have a child with a disease that is potentially curable with cord blood transplantation and are expecting another baby [8–10]. We report our experience on directed sibling cord blood banking for patients with β-thalassemia major, the likelihood of using a matched cord blood collection either alone or in combination with bone marrow from the same donor, and the outcome of such transplants in Greece. Patients and methods Eligibility for directed sibling cord blood banking, written consent and cord blood collection Families who already had a child with β-thalassemia major candidate for stem cell transplantation and expecting the birth of a healthy
108
E. Goussetis et al. / Blood Cells, Molecules, and Diseases 44 (2010) 107–110
Table 1 Laboratory characteristics of banked sibling cord blood units.
Volume in ml (w/35 ml anticoagulant) Nucleated cell count × 108 Total CD34+ cells/collection × 106 (n = 28) Viability %
Mean
SD
Range
98.9 7.8 2.8 98.6
31.1 5.1 1.9 1.6
(40–150) (2–20) (0.8–8.4) (88–100)
sibling were eligible for cord blood collection. Families were enrolled in an approved research protocol; written informed consent was obtained from the donor mother for screening of both the mother and the cord blood for mandatory microbiological markers, for tissue typing, and for the storage of the cord blood units. Families were not charged for cord blood banking. Enrollment began May 01, 1994. Characteristics of the banked cord blood units are shown in Table 1. The cord blood was collected before delivery of the placenta using a 250-ml blood bag with 35 ml citrate-phosphate-dextrose anticoagulant. Cord blood units were considered suitable for banking based on a net volume of ≥40 ml since even for low volume collections there is the possibility of combining the cord blood unit with a small volume of bone marrow from the same donor. Blood samples for HLA-typing, blood grouping, testing for infectious and genetic diseases, and for total and CD34+ cells counting were removed. All cord blood units collected between 1994 and 2003 (n = 38) were processed without volume reduction and stored in the bone marrow processing laboratory of the Stem Cell Transplantation Unit at Saint Sofia Children's Hospital. Cryoprotectant solution was prepared from dimethyl sulfoxide (DMSO) and 5% (wt/vol) human albumin solution. An equal volume of cooled 20% (vol/vol) DMSO solution was added to the cord blood. Cord blood cells were cryopreserved in freezing bags (Gabro, Haemofreeze, Amsterdam) using an automated, controlled cell freezer (Cryo10, CryoMed). In 2004, governmental funding facilitated the establishment of the first national cord blood bank, where 12 additional cord blood units were processed and stored as described by Rubinstein et al. [11]. Briefly, cryoprotectant solution was prepared from dimethyl sulfoxide and 10% (wt/vol) dextran-40/ saline solution. An equal volume of sterile, cooled 20% (vol/vol) DMSO solution was added to the cord blood in a controlled manner. The cord blood units after completion of the freezing program were stored in the liquid phase of a liquid nitrogen freezer at − 196 °C. Aliquots were taken post-processing for blood culture testing (1.0 ml for aerobic and anaerobic culture) with an incubation period of 7 days. Patients, grafts and conditioning regimens Our patients were classified in 1 of 3 classes according to the Pesaro criteria proposed by Lucarelli et al. [12]. Risk factors were hepatomegaly, portal fibrosis in the liver biopsy, and quality of
chelation therapy. Eight children with β-thalassemia major, 1 class I, 2 class II, and 5 class III, were transplanted by infusing either cord blood cells alone (n = 3) or cord blood cells combined with bone marrow cells (n = 5) from their HLA-matched siblings. Bone marrow cells were used because the number of cord blood NCs was considered insufficient to ensure engraftment according to current recommendations on cord blood transplantation for using a minimum NC dose of 4 × 107/kg for hemoglobinopathies [13]. Thus, following parental consent, we harvested a reduced bone marrow volume (median value 250 ml, range 190–280 ml) from the siblings aged between 2 and 3 years old. In our centre, for the transplantation of a thalassemic patient with a marrow graft, a minimum dose of 4 × 108 NCs/kg is required. The estimated bone marrow volume for this cell dose is approximately 500 ml for older patients with body weight N30 kg. Because cord blood was already available from the donors we proceeded with collecting less than half the volume required in case the transplant was to be performed solely with bone marrow graft, corresponding to a mean dose of 1.8 × 108 NCs/kg. Six cord blood units were thawed in a 37 °C waterbath with gentle agitation and immediately infused into the patients without further processing. In 2 cases, due to a donor-recipient major ABO incompatibility, cord blood cells were separated into a low-density fraction using Ficoll– Hipaque (d = 1.077 g/ml) after thawing. For both these cases the decision of combining cord blood with bone marrow was already based on the initial cell counts and therefore we were not concerned about further cell losses because of the Ficoll separation. Patients, donors, and graft characteristics are shown in Table 2. Seven patients underwent transplantation after a preparative regimen including busulfan (16 mg/kg), cyclophosphamide (200 mg/kg) and antilymphocyte globulin pre-transplant (40 mg/kg). The remaining patient was given a conditioning regimen including busulfan, cyclophosphamide, anti-thymocyte globulin, and fludarabine. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine A from day −1 to 9 months and then tapered off over the subsequent 3 months. Myeloid engraftment was defined as the first of 3 consecutive days when the absolute neutrophil count was 0.5 × 109/ l or higher, and platelet engraftment as the time to reach a sustained platelet count of 20 × 109/l or higher in the absence of platelet transfusion for 7 consecutive days. Donor hematopoiesis was documented by PCR study of genetic polymorphism of short tandem repeats. Results All 50 cord blood units collected were banked. All banked cord blood units were free of bacterial contamination. HLA-A, HLA-B, and HLA-DR typing was performed serologically. DNA analysis was used only in one case because there was one mismatched antigen. Eleven cord blood units were HLA identical to the prospective recipient and
Table 2 Patients, grafts and transplant outcomes. Patient 1 2 3 4b 5b 6b 7 8
Age 3 6 3 5 14 15 15 7
Weight 13 20 13 16 62 50 56 20
Pesaro Class I III II II III III III III
HLA
CB-NCa
CB-CD34a
BM-NC
Outcome
id 1Ag mm id id id id id id
(×107/kg) 6.8 8 5.2 0.3c 0.4c 1.8 1.4 3.2
(×104/kg) ND ND 20 6 17 5 8 16
(×108/kg) – – – 0.7 1.6 1.7 1.2 3.8
ATF 14 y AR AR-TF ATF 13 y ATF 12 y ATF 11 y ATF 4 y ATF 3 y
CB: cord blood; BM: bone marrow; NC: nucleated cells; ND: not done; id: identical; 1Ag mm: 1 antigen mismatch; ATF: alive and thalassemia free; AR: alive with thalassemia after rejection; AR-TF: alive, thalassemia free after second transplant with BM; y: years post-transplant. a The cord blood nucleated cell (CB-NC) counts reported were performed post-thawing of the units. b Data from these recipients are previously published [19]. c The counts reported were performed after Ficoll separation.
E. Goussetis et al. / Blood Cells, Molecules, and Diseases 44 (2010) 107–110
one cord blood unit was mismatched for one HLA-A antigen. Out of 12 cord blood units with an acceptable HLA-match, 8 have been transplanted, one unit with a NC dose of 2 × 107/kg was not infused because a sufficient bone marrow graft (10 × 106CD34+ cells/kg) was given; and, three remaining cord blood units are planned to be given within the next 3 years. In three patients, class I, II, and III respectively according to the Pesaro classification, cord blood containing N5 × 107 NCs/kg was the sole stem cell source while in 5 patients (Pesaro class II or III) the infused grafts consisted of cord blood (after thawing and processing) and bone marrow, containing 1.4 ± 1.2 × 107 and 1.8 ± 1.1 × 108 NCs/kg, respectively. Among the 3 patients who received a cord blood allograft alone, two had HLA-identical donors and one had a donor mismatched at 1 HLA-A antigen. Only one child with Pesaro risk class I had a stable engraftment; the other two (including the patient with the 1 antigen mismatched graft) experienced primary graft failure. Both children, who failed to engraft, were re-transplanted with bone marrow at a later date from the same donor; one has been cured and the other one did not engraft and lives with thalassemia. In all 5 recipients of combined cord blood and bone marrow allografts, neutrophil engraftment occurred at a median of 18 days (range, 17–29) and platelet engraftment at a median of 24 days (range, 19–32) post-transplantation. Outcomes after transplantation are shown in Table 2. Discussion Since the beginning of our stem cell transplantation program for thalassemia patients in 1994, families with thalassemic children, who desire a new pregnancy, have been offered the possibility of collecting and subsequently using cord blood cells in transplantation. We enrolled 50 families in a national directed sibling cord blood banking program, and banked all cord blood units collected. Both volume and cell counts (nucleated cell and CD34+) of cord blood units are comparable to those reported by banks in United States and the United Kingdom involved in collection of cord blood units from siblings of patients with thalassemia [8,9]. Regarding the usage rate of cord blood units in stem cell transplantation, our results are also in line with the experience in those countries, confirming the very high utility rate of HLA-matched cord blood units. Walters et al. [14] described the experience in the United States, where 32/96 of the donor-recipient pairs with β-thalassemia (33%) were HLA-identical and 14 of them (44%) received cord blood transplantation either alone or in combination with bone marrow or blood progenitor cells (n = 4) from the same donor. Twelve out of fourteen patients survive diseasefree after transplantation. One patient died due to pulmonary toxicity and the other one had disease recurrence. Smythe et al. [9] have reported their 10-year experience with directed sibling cord blood banking in the National Blood Service in England and Wales. They have used 7 cord blood units out of 36 collected from families affected by β-thalassemia. All of their patients survived disease-free (one had graft rejection but engrafted after bone marrow transplantation from the same donor). Most cord blood units stored, however, share only one HLA-haplotype with the recipient and are unlikely to be used. Our policy is to advise the family to donate the mismatched cord blood unit to the unrelated cord blood units pool; otherwise we request that the unit is transferred to a private bank. Prenatal HLA typing would be a method of selecting pregnancies appropriate for banking [15]. Graft failure is a well documented complication after cord blood transplantation for β-thalassemia and the main risk factors are low cell dose, use of methotrexate, and inadequate immune suppression. It seems likely, from the results of stem cell transplantation for βthalassemia, that the high graft failure rate can be overcome by employing additional pretransplant immune suppression including fludarabin, anti-thymocyte globulin, and thiotepa. One recent study has reported no graft failure or rejection in 27 children with βthalassemia transplanted with a conditioning regimen consisting of
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busulfan, thiotepa and fludarabin without methotrexate for graftversus-host-disease prophylaxis [16]. In addition, results of a multicenter comparative study, published as an abstract, demonstrated a 5-year disease-free survival of 86 ± 5% after matched sibling cord blood transplantation in 48 patients with beta-thalassemia major vs. 91% ± 3% in 184 patients who received matching sibling bone marrow during the same period [17]. These data from both studies suggest that cord blood transplantation result in a similar outcome to bone marrow transplantation, the majority of patients transplanted with cord blood in all published studies were young children with goodrisk disease (Pesaro class 1). For high-risk patients (Pesaro class II and III) of older age, such as those in our study, the use of additional bone marrow stem cells has proven valuable, especially when the cellular content of the cord blood unit is judged insufficient to secure engraftment. In such cases, one may argue that it is quite possible to delay the transplant until the donor is old enough to donate sufficient number of bone marrow stem cells, thus making cord blood storage unnecessary. However, for older thalassemic recipients with high body weight, the advantage of combining cord blood with bone marrow cells is the reduction of the volume of bone marrow harvest required and avoiding a long delay for the transplantation of children with Pesaro risk class II or III who already have poor prognosis [18]. Reduced volume bone marrow collection not exceeding 15 ml/kg can be an attractive option for enhancing the stem cell number in the graft without placing the donor at the same risk as a standard bone marrow collection. Such an approach was effective and beneficial in our patients, three of whom weighed 62, 50, and 56 kg, respectively. Moreover, as we noted in a previous report, the evidence of possible hematopoietic reconstitution derived from cord blood cells, shown by elevated fetal hemoglobin (HbF) levels, could be the result of the cooperation between a small bone marrow inoculum and the cord blood cells [19]. Our results are in agreement with the recommendation of delaying cord blood transplantation until the HLA-identical donor is at least 2 years of age [20]. The use of combined cord blood and marrow cell grafts for Pesaro class II and III patients, who are in high risk of rejection, is reasonable where a transplantation using cord blood units with high cell counts (NCs N4 × 107/kg) as the only source of stem cells should be reserved for young children classified in the good risk group (Pesaro class I). The use of combined grafts has been reported previously, but the exact criteria for supplementing cord blood with bone marrow have not been established [21,22]. Despite the encouraging results reported to date, cord blood transplantation for thalassemia has several limitations. The usage rates of units collected in directed banking programs reported by us and others are within the range of 15–20%. In our hands the efficacy of using cord blood alone in high risk patients was not as high as previously reported with good risk patients. However we do believe that banking programs should be encouraged but efforts for acquiring better cell yields from cord blood unit collections need to be continued and strategies of facilitating engraftment need to be explored further. References [1] G. Lucarelli, M. Galimberti, C. Giardini, P. Polchi, E. Angelucci, D. Baronciani, et al., Bone marrow transplantation in thalassemia. The experience of Pesaro, Ann. N.Y. Acad. Sci. 850 (1998) 270–275. [2] S.E. Lawson, I.A. Roberts, P. Amrolia, I. Dokal, R. Szydlo, P.J. Darbyshire, Bone marrow transplantation for beta-thalassemia major: the UK experience in two paediatric centres, Br. J. Haematol. 120 (2003) 289–295. [3] E. Angelucci, D. Baronciani, Allogeneic stem cell transplantation for thalassemia major, Haematologica 93 (2008) 1780–1784. [4] G. La Nasa, F. Argiolu, C. Giardini, A. Pession, F. Fagioli, G. Caocci, et al., Unrelated bone marrow transplantation for beta-thalassemia patients: the experience of the Italian Bone Marrow Transplant Group, Ann. N.Y. Acad. Sci. 1054 (2005) 186–195. [5] D. Gaziev, M. Galimberti, G. Lucarelli, P. Polchi, C. Giardini, E. Angelucci, et al., Bone marrow transplantation from alternative donors for thalassemia: HLA-
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