Collection of Cord Blood Stem Cells for Transplantation in Thalassemic Patients

Transcription

1 Collection of Cord Blood Stem Cells for Transplantation in Thalassemic Patients Surapol Issaragrisil," Sanan Visuthisakchai," Yaowalak Tangnaitrisorana," Dasnayanee Chandanayingyong? Vinai Suvatte: Voravarn Tanphaichitf Pornpimol Ruengvuttilert,d Anong Piankijaguma "Chulabhorn Bone Marrow Transplant Center and Departments of "Medicine, btransfusion Medicine, 'Pediatrics, and "Obstetrics and Gynecology, Siriraj Hospital, Mahidol University, Bangkok, Thailand Key Words. Cord blood Stem cell Transplantation Thalassemia Abstract. Thalassemia is widely distributed throughout the world and is one of the major public health problems. The use of bone marrow transplantation, the only curative therapy for thalassemia, is limited because less than 30% of the patients have unaffected and HLA-identical siblings as donors. Cord blood stem cells, an alternative source of stem cells for transplantation, have been successfully transplanted into patients with several diseases after myeloablative therapy. Twenty cord blood samples from unaffected neonates whose siblings had severe thalassemia were collected. The median volume was 80 ml. The median number of cells and colony forming units-granulocyte-macrophage in cord blood was 9.2 x lo* and 3.4 x lo5, respectively. Four of 20 cord blood samples had HLA-matched to the affected siblings. One patient underwent cord blood transplantation with success; one patient is waiting for transplantation. Introduction Thalassemia, the most common single gene disorder, is widely distributed from the Mediterranean across the Middle East through Southern Asia to Southeast Asia [ 11. In the Mediterranean alone, there are more than 200,000 patients with homozygous P-thalassemia. With more accurate diagnostic techniques, it is at present realized that thalassemia is more widespread than formerly recognized. There are at least Correspondence: Dr. Surapol Issaragrisil, Division of Hematology, Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Received August 9, 1995; accepted for publication August 9, OAlphaMed Press /95/$5.00/ million people carrying thalassemia genes. Recent migrations of people disperse thalassemia genes around the world. Refugees from Cambodia, Laos and Vietnam have brought the thalassemia genes to the USA and Europe. Thalassemia is therefore one of the major public health problems in the world. In Thailand, both thalassemias and hemoglobinopathies are prevalent [2, 31. The frequency of the a-thalassemia is 30-40% and the P-thalassemia occurs at a frequency of 3-9%. Hemoglobin E, the hallmark of Southeast Asia, is found with the frequency of 13%. There is a wide spectrum of manifestations of thalassemia syndrome varying from normal clinical and hematologic findings on one end to the severe forms on the other. Among 60 different clinical syndromes, homozygous P-thalassemia and P-thalassemia/hemoglobin E disease are the most common and severe clinical syndromes with which patients can be born. Control of thalassemia comprises prevention of the new births of thalassemic patients and provision of the best possible therapy to existing patients. Preventive measures include genetic counseling and prenatal diagnosis with induced termination of affected pregnancies. Hypertransfusion and iron chelation can improve life expectancy and quality of life, and can prevent undergrowth and thalassemic faces [4]. However, hypertransfusion is usually associated with high risk of blood-transmitted infections, especially HIV infection. Iron chelator is expensive and requires continuous subcutaneous infusion for at least 12 hours daily; therefore, patient compliance is frequently poor.

2 12 Cord Blood Transplantation in Thalassemia Bone marrow transplantation was first used to cure thalassemia in 1982 by Thomas and colleagues [5]. It is currently the only curative therapy for thalassemia [6-91. However, only less than 30% of the patients have unaffected and HLA-identical siblings to serve as donors. Moreover, a proportion of families have only the affected child. Experiences with transplants from mismatched related donors and unrelated donors are limited and the results are unsuccessful [lo]. Cord blood obtained at birth contains substantial numbers of hematopoietic progenitor cells [ 111, and may be used as an alternative source for transplantation [12]. A number of cord blood transplants have been performed [ We first reported the success of transplantation of cord blood stem cells from an unaffected, HLA-identical sibling into a patient with severe thalassemia [ 181. Materials and Methods Pregnant women whose children had had severe thalassemia underwent prenatal diagnosis during the first trimester. Prenatal diagnosis for thalassemia was performed using DNA extracted either from the chorionic villi or amniotic fluid fibroblast during the first trimester of pregnancy [19]. DNA was tested for the P-thalassemia mutation and the gene for hemoglobin E by hybridizing the amplified DNA with horseradish peroxidaselabeled antistreptolysin 0 and pe probes, respectively. If the fetuses were unaffected, the pregnancy was then continued. DNA typing for HLA class I1 antigens was performed with the UCLA technique involving sequence-specific primers [20]. After delivery, cord blood was collected into tubes containing acid citrate dextrose (ACD) or heparin with minimal essential medium. There was no attempt at cell separation; whole cord blood was frozen in 10% dimethylsulfoxide and kept in liquid nitrogen. Cell count, progenitor cell assay, HLA-A, -B, -DR typing and bacterial culture were performed. Results During the period from May 1993 to July 1995, 20 cord blood samples from unaffected Table I. Cord blood stem cell collection-siriraj experience Number 20 Volume 80 (40-130) ml Cells 9.2 x lo8 ( x lo*) CFU-GM 3.4 x 105 ( x 105) children were collected. ACD was used as anticoagulant in five samples. After having partial clot in one sample, we changed the anticoagulant to heparin. The median volume was 80 ml (range ml) (Table 1). The median number of cells and colony forming units-granulocyte-macrophage (CFU-GM) in cord blood was 9.2 x lo* (range x lo8) and 3.4 x lo5 ( x lo5), respectively. The majority of samples was sterile. One child died of septicemia nine hours after delivery; his cord blood was positive for bacillus and was discarded. One cord blood sample was clotted due to inadequate anticoagulant. One sample was contaminated with bacteria. Of 20 cord blood samples collected, only four had HLA matched to the affected siblings (Table 11). One patient underwent cord blood transplantation with success [ 181. Patient data are summarized in Tables I11 and IV. One patient is waiting for transplantation. The other two patients were not transplanted because cord blood was clotted in one patient and was contaminated with bacteria in the other. Discussion Curative therapy for thalassemia using HLAidentical sibling marrow grafts is limited because only less than 30% of the patients have an unaffected and HLA-identical sibling as a donor. Moreover, a proportion of the families have only the affected child; they don t want to risk Table 11. Cord blood stem cell collection and transplantation-siriraj experience Number 20 HLA-incompatible 16 HLA-matched 4 - Transplantation 1 - Waiting for transplantation 1 - Partially clotted cord blood 1 - Bacterial contamination 1

3 Issaragrisil et al. I3 Table 111. Pretransplant data of cord blood transplantation in thalassemia Patient Age Sex Diagnosis Previous blood transfusion Iron chelation Serum Ferritin Conditioning (GVHD) prophylaxis Blood group HLA typing Donor after Transplantation Sex Male Blood group 0 positive HLA typing identical Cord blood volume 44 ml Cells 5.46 x lo8 CFU-GM 2.29 x years Female P-thalassemia/hemoglobin E disease 18 units yes 190 ng/ml (normal ) Busulfan 14 mg/kg Cyclophosphamide 200 mg/kg Short methotrexate Cyclosporine 0 positive A2.2; B46 (Bw~), B46(Bw6); DRB1*0403, *0803; DQ B1*0302, *05 having more births of thalassemic children. Previous results indicate that transplants from mismatched related donors and unrelated donors were not successful [lo]. The use of mismatched related and matched unrelated transplantations is therefore limited. Prenatal diagnosis has made the new births of unaffected children possible. They can serve as donors for marrow transplantation when their HLA antigens are identical to their affected siblings. However, a newborn is not an appropriate donor; the youngest donor ever used was reported to be six months old [21]. Bone marrow Table IV. Transplant and post-transplant course Date of Transplant Cell dose/kg CFU-GM/kg G-CSF therapy WBC > 1.O x 10 L ANC > 0.5 x 1Oy1L WBC > 3.0 x ioy/l ANC > 1.O x 1OY/L Platelet > 20 x 1 Oy/L Platelet > 50 x 10y/L GVHD Chronic GVHD Disease-free survival June 12, x x lo4 Yes Day 23 Day 23 Day 53 Day 53 Day 31 Day 44 No No 48 months transplantation must therefore be postponed until the donors are grown. Allogeneic bone marrow transplantation has been successfully used in thalassemic patients, particularly those in Lucarelli s class 1 (no hepatomegaly and no portal fibrosis). The risks are greater for those who have liver damage due to iron overload and associated viral hepatitis (classes 2 and 3); the rejection rate is high. Our experience is in accordance with Lucarelli s results. Early bone marrow transplantation offers a very high probability of cure. In those who have full-blown manifestations with chronic anemia, hepatosplenomegaly and marked erythroid hyperplasia in the bone marrow, the success rate is low due to a high rate of graft failure. Practically, the severity of the disease should be determined based on genotype, and bone marrow transplantation should be performed as soon as possible in children with severe disease. Cord blood is an alternative source of stem cells for transplantation. According to the International Cord Blood Transplant Registry [22], 50 children underwent cord blood transplantation; 20 nonmalignant diseases and 30 malignant diseases. The median age of the patients was 4.7 years. The body weight was from kg (median = 19 kg).

4 74 Cord Blood Transplantation in Thalassemia Thirty-nine sibling donor recipient pairs were HLA-identical. Eleven were HLA-mismatched (one antigen in four, two antigens in one and three antigens in six). Donor age varied from weeks (median 39 weeks). The volume of cord blood collected was 100 ml (range ml). The numbers of cells and CFU-GM in the graft were 2-73 x lo8 (median = 7.8 x lo8) and x IO (median = 4.2 x lo5), respectively. The median numbers of cells and CFU-GM in the graft on the basis of the patient s body weight were 5 x lo7 and 1.9 x lo4, respectively. Engraftment after cord blood transplantation was evident in 90% of the patients. However, neutrophil and platelet recovery were delayed after cord blood transplantation compared to bone marrow transplantation. The median time of neutrophil and platelet recovery was 22.5 days and 49 days after transplantation. There was no correlation between nucleated cell and progenitor cell dose and hematopoietic recovery. The use of hematopoietic growth factor did not shorten the time to neutrophil recovery. Failure to engraft occurred in 7 of 50 patients; 6 in nonmalignant diseases. Graft-versus-host-disease (GVHD) occurred infrequently and was found only in recipients of HLA-antigen-mismatched transplants. With regard to unrelated cord blood transplantation, 2 1 patients (acute lymphoblastic leukemia, 10; acute inyeloid leukemia, 6; chronic myelocytic leukemia, 1 ; Fanconi s anemia, 2; severe aplastic anemia, 1 and other, 1) received cord blood stem cells from an unrelated donor [22]. HLA antigens were identical in three and mismatched in 18 (for one antigen in 13, two antigens in four and three antigens in one). Four patients died before day 28 after transplantation. Two patients had no evidence of engraftment. Fourteen patients survived with long-term disease-free survival. Acute GVHD was found in 12 of 15 evaluable cases. Results using mismatched related and unrelated cord blood transplants demonstrated that engraftment is feasible and that the degree of GVHD from unrelated and haploidentical cord blood may be less than that seen with similarly mismatched bone marrow. We first reported the successful use of cord blood transplantation in a patient with severe thalassemia [ IS]. Neutrophil and platelet recovery was slower compared to bone marrow transplantation [ IS]. Engraftment was evident although there was still some host hematopoiesis. Recently, there was another case report of cord blood transplantation in a patient with thalassemia. Unfortunately the patient had no evidence of engraftment [22]. There are limited data on cord blood transplantation in thalassemic patients. Further clinical transplantation studies in a larger number of individuals will be needed to determine the efficacy of cord blood transplantation in thalassemic patients. Acknowledgments This work was supported by the Chulabhorn Foundation, the Siriraj-China Medical Board, the State of Baden Wiirttemberg, Germany, the Daimler Benz Company, the Volkswagen Foundation and the National Science and Technology Development Agency, Thailand. S. Issaragrisil is a recipient of the Senior Fellowship Award from the National Science and Technology Development Agency, Thailand. References Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. Oxford: Blackwell Scientific, 1981: Wasi P, Piankijagum A, Issaragrisil S. Geographical variation in blood disease: Southeast Asia. In: Weatherall DJ, Ledingham LGG, Warrell DA, eds. Oxford Textbook of Medicine. Oxford: Oxford Medical Publications, 1987: Wasi P. Haemoglobinopathies including thalassaemias. Part 1: tropical Asia. Clin Haematol 1981 ;10: Piomelli S. Cooley s anemia management: 25 years of progress. In: Buckner CD, Gale RP, Lucarelli G, eds. Advances and Controversies in Thalassemia Therapy: Bone Marrow Transplantation and Other Approaches. New York: Alan R. Liss, 1989: Thomas ED, Buckner CD, Sanders JE et al. Marrow transplantation for thalassaemia. Lancet 1982;2: Lucarelli G, Galimberti M, Polchi P et al. Marrow transplantation in patients with advanced thalassemia. N Engl J Med 1987;316: SS. 7 Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990;332: Lucarelli G, Galimberti M, Polchi P et al. Marrow transplantation in patients with thalassemia

5 Issaragrisil et al. 75 responsive to iron chelation therapy. N Engl J Med 1993;329: lssaragrisil S, Visuthisakchai S, Suvatte V et al. Bone marrow transplantation for thalassemia in Thailand. Bone Marrow Transplant 1993;12(suppl I ): Polchi P, Galimberti M, Lucarelli G. HLA-mismatched bone marrow transplantation in thalasseinia. Bone Marrow Transplant 1993; 12(suppl I ): Broxmeyer HE, Douglas GW, Hangoc G et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989:86: Broxmeyer HE, Kurtzberg J, Gluckman E et al. Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. Blood Cells 1991;17: Gluckman E, Broxmeyer HE, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical cord blood from an HLA-identical sibling. N Engl J Med 1989;321: Vilmer E, Sterkers G, Rahimy C et al. HLA-mismatched cord blood transplantation in a patient w it h ad v a n c ed 1 e u ke mi a. Tr an s pl an t a t i o n 1992~53:1155-1l57. IS Wagner JE, Broxmeyer HE, Byrd RL et al. Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood 1992;79: I. 16 Vowels MR, Lam-Po-Tang R, Berdoukas V et al. Correction of X-linked lymphoproliferative disease by transplantation of cord blood stem cells. N Engl J Med 1993;329: Wagner JE. Umbilical cord blood transplantation: overview of the clinical experience. Blood Cells 1994;20: Issaragrisil S, Visuthisakchai S, Suvatte V et al. Transplantation of cord-blood stem cells into a patient with severe thalassemia. N Engl J Med 1995:332: Winichagoon P, Fucharoen S, Siritanaratkul N et al. Prenatal diagnosis for P-thalassemia syndromes using HRD-labelled oligonucleotide probes at Siriraj Hospital. Southeast Asian J Trop Med Public Health 1995;26(suppl 1): Park MS, Tonai R. Phenotype frequencies of the class I1 (DR, DQ) DNA alleles by the patterns of sequence-specific primer mixture (SSPM) in four different populations and the probable haplotypes between DR B1 allele and DQ B 1 allele. In: Terasaki PI, Cecka JM, eds. Clinical Transplants Los Angeles: The Regents of the University of California, 1993: Buckner CD, Clift RA, Sanders JE et al. Marrow harvesting from normal donors. Blood 1984;64: Wagner JE. Results of the International Cord Blood Transplant Registry. In: Joint International Workshop on Foetal and Neonatal Hematopoiesis and Mechanisms of Bone Marrow Failure, Paris, France, April 3-6, 1995 (in press).