Bone Marrow Transplantation (2004) 33, 33–38 & 2004 Nature Publishing Group All rights reserved 0268-3369/04 $25.00 www.nature.com/bmt

Cord blood stem cells Umbilical cord blood transplant for adult patients with severe aplastic anemia using anti-lymphocyte globulin and cyclophosphamide as conditioning therapy

P Mao, S Wang, S Wang, Z Zhu, Q Liv, Y Xuv, W Mo and Y Ying

Department of Haematology, First Municipal People’s Hospital, Guangzhou, China

Summary: therapy with immunosuppressive agents.1 For patients without a sibling donor having no response to one or Allo-CBSCT (cord blood stem cell transplant) has more courses of immunosuppressive therapy a fully been applied in sixadult patients with severe aplastic matched unrelated donor BMT should be considered anemia (SAA). Anti-lymphocyte globulin (ALG) asalternative salvagetherapy. Finding related or unrelated 40 mg kgÀ1 dÀ1 Â 3 days combined with cyclophosphamide individualswho are HLA-identical to some (CTX) 20 mg kgÀ1 dÀ1 Â 3 days constituted a lower patients, however, is difficult and time consuming because intensive conditioning regimen. The prophylaxis of of the extreme polymorphism of most HLA GVHD consisted of standard CsA and MTX. Patients loci. Umbilical cord blood isanother alternative sources are all male having a mean age of 26.5 years (range 22– of stem cells that improves donor availability for trans- 38), and a median weight of 55.6 kg (range 52–60 kg). plantation because frozen and stored UCB can be Cord blood searches were all conducted at Guangzhou made available on demand. Infectiousagents,particularly Cord Blood Bank. Three of sixpatients in our study cytomegalovirus(CMV), are rarely seenin the new received one unit of cord blood in a procedure, whereas for born than in adults. Furthermore, UCBT may have a another three patients, two units of cord blood (double lower incidence and severity of GVHD than conventional units) were infused at the same time in a transplant BMT, allowing successful transplantation in the HLA- protocol. The nine units of umbilical cord blood (UCB) mismatched recipient. Recently, investigators have ex- infused contained 1.6–10.7 Â 107 nucleated cells/kg body plored the applicability of UCB stem cells as a rescue weight of the recipient after thawing. HLA antigens were therapy for both malignant and non-malignant hematolo- identical in one unit, 1 antigen mismatched in seven, 2 gical diseases with some promising results.2,3 We first antigens mismatched in 1. As of February 2003, after a reported in 2000 an UCB transplantation from unrelated median follow up of 20 months (range 7–50), four patients donor in an adult with SAA4 and now report additional are alive and disease free. Five patients engrafted with experience. molecular biology analyses showing donor-recipient mixed chimerism post transplant which is stable and persistent. One patient died of severe infection in the third month from transplant and another patient died in the early stage Patients and methods post transplant of serious aspergillus infection without evidence of engraftment. Patients Bone Marrow Transplantation (2004) 33, 33–38. Six adult patientswith SAA on our studyreceived allo- doi:10.1038/sj.bmt.1704295 CBSCT from December 1998 to January 2002. All patients Keywords: umbilical cord blood; stem cell transplanta- had previously failed immunosuppressive therapy and had tion; severe aplastic anemia no HLA-matched sibling donor. They were all male with the average age of 26.5(22–38) at the time of transplant. Mean interval from diagnosis to CBSCT was 4.6 months (range 2–10 months) and all remained transfu- BMT remainsthe firstchoice in children and young adults sion-dependent when transplanted. All patients were with SAA. Full matched sibling BMT may also considered transfused before transplant and all but one had more in older patients if there is no response to the first-line than four transfusions. Bone marrow smears and biopsy showed failure of hematopoiesis and increased nonhema- topoietic cells and fat cells. Hemolytic screening tests were negative for acidified-serum hemolysis, sucrose lysis test Correspondence: Dr P Mao, First Municipal Peoples Hospital of Guangzhou, Haematology Department, 602, Renmin road, Guangzhou, and venom hemolysis test. In all patients, severe aplastic 5 510180, China; E-mail: [email protected] anemia was diagnosed by standard criteria with the Received 08 April 2003; accepted 05 July 2003 exclusion of MDS. Umbilical cord blood transplant for adult patients P Mao et al 34 Umbilical cord blood according to blood CsA level and MTX 15 mg/m2 (day 1) and 12 mg/m2 (days3, 6, 11). They alsoreceived UCBswere found in Guangzhou cord blood bank. They methyprednisolone 1.0 mg kgÀ1 dayÀ1 p.o. from day 1 to were collected after delivery and cryopreserved in 10% 14, 0.5 mg kgÀ1 dayÀ1 from day 15 to day 28. DMSO in a programmed cell freezer to avoid cell loss which might impair engraftment. Syphilisand viral testsincluding HIV, hepatitisB and C and CMV were performed on both Supportive care mothers’ blood and cord blood units. Tests for genetic diseases such as thalassemia were also performed. Each cord Prophylaxisof infectionsincluded acyclovir, fluconazole, blood waskept in liquid nitrogen below À1921C. Trypan ofloxacin, metronidazole and SMZ. CMV-seropositive blue test to determine the viable cell frequency, flow patients(patients1, 2, and 5) received prophylactic cytometry to count CD34 þ cellsand cell culture for ganciclovir. All blood productswere irradiated and filtered. CFU-GM were done immediately after the cellswere Red blood cell and platelet transfusions were performed to thawed. UCBsrecovery rate were 79–93.5% after thawing. maintain a hemoglobin of 470g/l, platelet count 9 All UCBsand related patientsdata were listedin Table 1. 420 Â 10 /l. Patientswere given a combination of G-CSF 5ugkgÀ1 dayÀ1 and EPO 6000 IU every other day from day 0 until recovery from aplasia. All patients received Preparative conditioning regimen intravenousimmunoglobulin prophylaxisand hyperali- Patients in our study received a pretransplant regimen of mentation when needed. both CTX 20 mg/kg/day i.v. on days-6, -5, -4, and ALG À1 À1 40 mg kg day i.v. on days-3, -2, -1. CTX wasgiven Assessment of engraftment daily as a 2 h infusion. Mesna was administered for uroprotection before the first dose of CTX to 24 h after Hematopoietic chimerism was evaluated by cytogenetic the last dose of CTX. ALG was given daily by continuous methodsasmicrosatelliteDNA fingerprinting for detection i.v. infusion over 6 h. of multiple short tandem repeat loci. The DNA samples were extracted from bone marrow and /or peripheral white GVHD prophylaxis blood cells. ABO-mismatched pairs were usually tested to find conversion of the blood group. All patients received GVHD prophylaxis consisting of CsA The immunoreconstitution evaluation was carried out 4 mg/kg/day (days1-5) followed by regulated dosage prior to and 1, 2, 3, 6, 9, 12 monthsafter transplantation

Table 1 Characteristics of patients, donors, and cord blood data after thawing

Patient Age Weight HLA-mismatched loci Sex ABO group MNC Â 107/kg CD34+ Â 105/kg CFU-GM Â 104/kg no. (kg) (donor/recipient) (donor/recipient) (donor/recipient)

1 23 60 — M/M AB/B 1.89 1.70 1.80 2 25 59 B13,DR1202/ M/M B/A 10.7 17.20 13.50 B61(40),DR0402 3 26 60 A11/A30(19) M/M O/B 1.60 1.22 1.63 4 37 50.5 A24/A1102 M/M O/B 2.43 1.80 2.96 DR03/DR0403 M/M O/B 3.26 4.43 3.20 5 22 56 B13(Donor1)/B75(15) F/M A/O 2.56 2.94 1.69 B46(Donor2)/B39(16) F/M AB/O 2.18 1.16 1.96 6 24 52.5 B38(16)/B51(5) M/M B/A 4.42 6.74 8.16 B38(16)/B46 F/M A/A 2.20 1.49 2.14

M ¼ male; F ¼ female. Patients 4–6 receiving double-unit transfusion.

Table 2 Consecutive observations for recipient’s peripheral blood

The month WBC( Â 109/L) BPC( Â 109/L) Hb(g/L) post transplant Patient1 Patient 2 Patient 4 Patient 5 Patient 1 Patient 2 Patient43 Patient54 Patient 1 Patient 2 Patient43 Patient54

0.5 2.6 0.8 1.0 1.2 31 19 16 18 73 62 36 46 1 3.7 2.1 2.1 1.8 62 13 46 20 89 65 32 78 3 4.0 2.4 3.2 2.7 131 21 80 49 123 58 66 126 6 4.1 2.5 3.0 3.6 125 28 82 42 119 86 76 120 9 3.9 4.0 4.2 3.7 161 36 118 68 143 107 90 152 12 4.4 5.8 5.0 5.2 147 88 196 102 135 145 116 159 24 4.8 5.5 186 261 146 142 36 4.6 154 141 48 6.2 184 148

Bone Marrow Transplantation Umbilical cord blood transplant for adult patients P Mao et al 35 and subsequently every 6 months for those longer surviving cytes, T-lymphocytes recovered to normal levels after 6 patients. The investigated immunological parameters in- months. CD4 þ T-lymphocytes were decreased signifi- cluded lymphocyte count, B-lymphocytes, T3-, T4-, T8- cantly during the first 150 days posttransplant while lymphocytes, T4/T8 ratio, natural killer cell (CD3À CD16 þ CD56 þ ) activity and serum-IgG, -IgA, IgM.

Results

Engraftment and outcome Evidence of engraftment wasfound in five patientsby molecular biology analyses showing donor-recipient mixed chimerism post transplant which is stable and persistent. Median time to ANC 40.5 Â 109/L was18.4 days(range 15–25 days); the median time for platelets counts 420 Â 109/L was37.7 days(range 15–79 days).One patient died of severe infection (both staphylococcemia and trichomycosis nigra) in the third month from transplant though there wasevidence of engraftment. Another patient also died of disseminated aspergillus infection in the early stages post transplant without engraftment. No patient developed CMV disease after transplant. As of February 2003, after a median follow-up of 20 months(range 7–50 months), four patients are alive and disease free. Their peripheral blood counts are shown in Table 2. These four patients have become transfusion-independent and achieved hematopoietic reconstitution after the transplant. The bone marrow aspirates had demonstrated hematopoie- tic aplasia until 2, 4, 2.5, and 3 months post transplant and showed complete recovery of hematopoiesis for 13.5, 16, 17, and 18.5 months observed from the transplant in patients1, 2, 4, and 5, respectively. NK cell activity generally recovered around 50 days post transplant. Absolute lymphocyte count, B-lympho-

Figure 1 Microsatellite DNA fingerprinting for patient 1. Lane A, marker; lane B, cord blood; lane C, the recipient before transplant; lane Figure 2 Fluorescent detection of multiple short tandem repeat loci for D, 3 months after transplant; lane E, 6 months after transplant; lane F, 12 patient 2. Marker 15 wasthe symbolindicating the cellsfrom the donor months after transplant; lane G, 36 months after transplant. engrafted into the recipient.

Bone Marrow Transplantation Umbilical cord blood transplant for adult patients P Mao et al 36

Figure 3 Successful engraftment of single unit of cord blood indicated by DNA fingerprinting (patient 5) Markers 32 , 9, 15.2 appeared in each correspondent Loci were symbols indicating the cells from donor 2 engrafted into the recipient (patient 5).

CD8 þ T lymphocytesincreasedto valueshigher than serum-IgG, IgA, IgM levels decreased persistently during normal up to 5 months. Consequently the T4/T8 ratio the initial 3 monthsand recovered gradually to normal 9, decreased and continued below 0.9 until 24 months. The 19, 5 months after transplantation, respectively.

Bone Marrow Transplantation Umbilical cord blood transplant for adult patients P Mao et al 37 Donor –recipient chimerism receiving 41.0  107 NC/kg in UCBT including some adult patients. Kai8 reported six adults with leukemia receiving Repeated DNA amplification and microsatellite DNA UCBT and all the patientshad full chimerism.We report fingerprinting showed persistent stable chimerism in those here durable donor-recipient mixed chimerism can be four patients(patients1, 2, 4, and 5). The studyof patient 1 achieved by unrelated UCBT in adult patientswith SAA, for a long period has demonstrated a stable mixed suggesting again that UCB could be employed as a source chimerism post transplant (Figure 1). The chimerism of hematopoietic stem cell for adult transplantation. analyses of peripheral blood and bone marrow in patient In our study durable and stable donor-recipient mixed 2 revealed 40% and 42% donor hematopoiesis 3 and 12 chimerism was formed after transplant to provide a months after transplant, respectively (Figure 2). In patients platform at which the immunotolerance may be created (patients4 and 5) receiving two unitsof cord blood, only a between the cellsof donor and recipient. Patientsare single unit engrafted in each of them (Figure 3). DNA of usually prepared for transplantation with cyclophospha- the donor cellsfirstappeared in bone marrow and/or mide of total dose 200 mg /kg,9 whilst in our group, only peripheral blood samples in these two patients around day one third of that dose was used in the preparative 20, when the leukocyte count wasjustabove 1.0 109/L. þ  conditioning regimen. The combination of ALG and Thereafter, and from a few daysbefore the leukocyte count CTX we use is enough to suppress the T-lymphocytes of increased to around 3.0 109/L, donor cellswere obviously  the host and to facilitate engraftment of UCB cells demonstrated in the recipients by short tandem repeat loci producing stable donor-recipient mixed chimerism. This detection and a stable mixed chimerism with 30–40% regimen may also reduce transplantation related toxicity. donor hematopoiesis has long been observed. In another We transplanted SAA patients with UCB units mis- patient (patient No. 6) who received two units, no evidence matched with the recipient for 1 (n ¼ 2) or 2 HLA loci of engraftment of either unit wasfound. ABO blood group (n ¼ 1). Cell dose was the most important factor for changed from host origin into donor type in two patients engraftment and survival.10,11 Double chimerascreated by (patients2 and 5) developing 2 and 2.5 monthspost UCBT from two partially matched unrelated donorshave transplant respectively. been recently reported.12 Two unitsof UCB cellstrans- planted into a recipient may increase the cell dose to GVHD support reconstitution of hematopoiesis especially in the early stage of transplant. We have two patients receiving Chronic GVHD wasdiagnosedin two patientslimited to double unitsof UCBT but resultingin engraftment of only the skin, developing after withdrawal of CsA. Localized one unit. It remainsunclear whether double unit trans- erythematousplaquesand papuleswere manifested.The plantation hasthe advantage of increasingengraftment skin biopsy demonstrated a subcutaneous infiltration by over one unit. lymphocytes. No treatment has been undertaken for this We summarize that unrelated umbilical cord blood could mild cGVHD that has been persistent but has not be employed as a source of hematopoietic stem cell for progressed. There has been no evidence of GVHD in the adult patientswith SAA. Our ALG and CTX based gut or in the liver. conditioning regimen proveseffective, well tolerated, less toxic, and safe. The following use of immunosuppressive agents like CsA may also exert a therapeutic effect in Discussion addition to transplant.

Umbilical cord blood hasrecently emerged asan alter- native source of hematopoietic stem cells especially for References those patients lacking an HLA-matched related donor. Cord blood can be collected and stored on a large scale, has 1 Schrezenmeier H, Bacigalupo A, Aglitta M et al. Guidelines no risk to donors, a lower risk of GVHD and the ability to for treating aplastic anemia. In: Schrezenmeier H , Bacigalupo Aplastic anemia reconstitute hematopoiesis and immunity after transplant. A (ed.) . Cambridge University Press: Cam- bridge, p. 308. UCB containsa significantlyhigher number of early and 2 Gluckman E. European results of unrelated cord blood committed progenitor cellsand now iswidely usedin transplants. Bone Marrow Transplant 1998; 21 (suppl 3): patientswith malignant and non-malignant hematological 587–591. diseases. UCBT mainly remains applied to children because 3 Wagner JE, Rosenthal J, Sweetman R et al. Successful of the limited number of hematopoietic cells, raising the transplantation of HLA-matched and HLA-mismatched um- question of whether it can reconstitute adult patients. bilical blood from unrelated donors: analysis of engraftment Umbilical cord blood stem cell transplantation has been and acute graft-versus-host disease. Blood 1996; 88: 795–802. successfully employed in our study for an adult patient with 4 P Mao, C Liao, Z Zhu et al. Umbilical cord blood SAA since 1998.6 The patient remained in a complete transplantation from unrelated HLA- matched donor in an Bone Marrow Transplant hematopoietic recovery for 50 months. Cytogenetic evi- adult with severe aplastic anemia. 2000; 26: 1121–1123. dence showed long term stable donor-recipient chimerism. 5 Camitta BM. Criteria for severe aplastic anemia. Lancet 1988; UCB transplant has been performed for additional SAA 1: 303–304. adult patients thereafter and the similar successful engraft- 6 Gluckman E. Use of hematopoietic stem cells from cord blood ment of UCB cellscould be reproduced. Gluckman 7 for allogeneic transplantation in man. Bull Acad Ndt Med indicated that neutrophil recovery was76% in patients 1998; 182: 337–348.

Bone Marrow Transplantation Umbilical cord blood transplant for adult patients P Mao et al 38 7 Gluckman E, Rocha V, Chevret S. Results of unrelated marrow transplantation for adult patients with acute leukemia umbilical cord blood hematopoietic stem cell transplant. in complete remission. Br J Haematol 2002; 118: 140–143. Transfus Clin Biol 2001; 8: 146–154. 11 Kurtzberg J, Langhlin M, Graham ML et al. Placental blood 8 Kai S. Cord blood transplantation for adults. Rinsho Byori as a source of hematopoietic stem cells for transplantation into 1999; 110: 48–53. unrelated recipients. N Engl J Med 1996; 335: 167–170. 9 Sanz GF, Saavedra S, PlanellesD et al. Standardized, 12 Barker JN, Weisdorf DJ, Wagner JE. Creation of a double unrelated donor cord blood transplantation in adults with chimera after the transplantation of umbilical-cord blood from hematologic malignancies. Blood 2001; 98: 2332–2338. two partially matched unrelated donors. N Engl J Med 2001; 10 Ooi J, Iseki T, Takahasi S et al. A clinical comparison of 344: 1870–1871. unrelated cord blood transplantation and unrelated bone

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