Cord blood stem cells Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood transplantation

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1 (2004) 33, & 2004 Nature Publishing Group All rights reserved /04 $ Cord blood stem cells Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood transplantation AP Iori 1, R Cerretti 1, L De Felice 1, M Screnci 1, A Mengarelli 2, A Romano 2, M Caniglia 3, L Cerilli 1, G Gentile 1, ML Moleti 1, F Giona 1, F Agostini 1, I Pasqua 1, MP Perrone 1, MR Pinto 3, L Grapulin 4, AM Testi 1, P Martino 1, G De Rossi 3, F Mandelli 1 and W Arcese 1 1 Department of Cell Biotechnology and Hematology, University La Sapienza, Rome; 2 Istituti Fisioterapici Ospitalieri, Istituto Regina Elena, Rome; 3 Bambino Gesù Hospital, Division of Pediatric Hematology, Rome; and 4 Institute of Radiotherapy, University La Sapienza, Rome Summary: From July 1995 to December 2001, 42 patients with leukemia aged 1 42 years underwent cord blood transplant (CBT) from unrelated, p2 antigen HLA mismatched donors. In all, 26 patients were in p2nd complete remission and 16 in more advanced phase. Conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policy were uniform for all patients. The cumulative incidence of engraftment was 90% (95% CI: 8 1). The cumulative incidence of III IV grade acute- and chronic-gvhdwas 9% (95% CI: 4 4) and 35% (95% CI: 1 0), respectively. The 4-year cumulative incidence of transplant-related mortality (TRM) and relapse was 28% (95% CI: 7 7) and 25% (95% CI: 4 5), respectively. The 4-year overall survival (OS), leukemia-free survival (LFS) and event-free survival (EFS) were 45% (95% CI: 7 3), 47% (95% CI: 0 4) and 46% (95% CI: 0 2), respectively. In multivariate analysis, the most important factor affecting outcomes was the CFU-GM dose, associated with CMV serology (P ¼ 03 and 4, respectively) in influencing OS and with patient sex (P ¼ 08 and 3, respectively) in influencing LFS. Finally, CFU-GM dose was the only factor that affected EFS significantly (P ¼ 2). In conclusion, the infused cell dose expressed as in vitro progenitor cell growth is highly predictive of outcomes after an unrelated CBT and should be considered the main parameter in selecting cord blood units for transplant. (2004) 33, doi:1038/sj.bmt Published online 19 April 2004 Keywords: cord blood transplant; leukemia; unrelated HSC transplant; prognostic factors Correspondence: Prof W Arcese, Cattedra di Ematologia, Dipartimento di Biopatologia e Diagnostica per Immagini, University of Rome Tor Vergata, Via Montpellier, 1, Rome, Italy; william.arcese@ptvonline.it Received 13 October 2003; accepted 4 February 2004 Published online 19 April 2004 Cord blood represents the most recent source of hematopoietic stem cells used in the unrelated transplant setting for patients who lack a related HLA compatible donor Two comparative studies have shown that, in children, unrelated cord blood transplant (CBT) is an acceptable alternative to unrelated bone marrow (BM) transplant. 13,14 Furthermore, three recent reports from either multicentric 9 or unicentric 10,12 experiences suggest that CBTcould be considered a reasonable alternative also in adults provided that an adequate number of nucleated cells (NC) is infused. Indeed, the number of NC has been identified in several reports as the most significant factor associated with rate of engraftment, transplant-related mortality (TRM) and survival, 1,3 5,7 9,15,16 while three studies reported the number of infused colony-forming cells (CFC) or CD34 þ cells, respectively, as better predictors than NC for engraftment and patient outcomes. 9,11,16 However, most of the abovementioned studies are retrospective, multicentric and include patients with heterogeneous diseases and short follow-up. Herein, we report long-term outcomes and pre-transplant prognostic factor analysis for 42 consecutive patients with high-risk leukemia prospectively undergoing an unrelated CBTin two Centers participating in the same transplant program. Patients and methods Eligibility criteria Searches for an unrelated hematopoietic stem cell donor were undertaken for all patients aged o45 years and lacking a related HLA compatible donor, if they met the following disease criteria: acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) in 2nd or subsequent complete remission (CR) or in relapse with o30% of blasts in the marrow; refractory anemia with excess of blasts (RAEB), chronic myeloid leukemia (CML) in 41st chronic phase (CP) and juvenile myelomonocytic leukemia (JMML). Patients with acute leukemia in 1st CR were also included if they had at least one of the following poor

2 1098 prognostic features: t(9;22) translocations or involvement of chromosome band 11q23, and molecular evidence (by RT-PCR) of persistent leukemia X3 months after CR; failure to achieve CR with primary induction regimen; AML diagnosed after an antecedent myelodysplastic syndrome. Patients with acute leukemia in 1st or 2nd CR at transplant were considered to have intermediate disease; patients with acute leukemia in relapse or 42nd CR and patients with chronic leukemia in 41st CP were considered to have advanced disease. HLA typing was determined molecularly for both class I (HLA-A, B, C) and class II (HLA-DR, DQ) antigens; DRB1 alleles were defined by high-resolution methods. A CB unit was considered suitable for transplant when p2 HLA antigen (HLA-A, B, DRB1) differences with the recipient were detected and its content of nucleated cells was at least /kg of the recipient body weight (bw). As rescue, all patients had an autologous cryopreserved unit of hematopoietic stem cells collected from marrow or peripheral blood as backup. Informed consent was required from the patient and/or guardian before CBT. Between July 1995 and December 2001, 42 consecutive patients with intermediate or advanced leukemia underwent CBTfrom an unrelated donor. Out of them, 14 have been previously reported. 6 Transplant procedures CB units were provided by the cord blood banks of GRACE, Italy (Milano, Roma, Torino, Firenze, Padova) (n ¼ 22), New York, USA (n ¼ 6), Du sseldorf, Germany (n ¼ 6), Barcellona, Spain (n ¼ 4), St Louis, USA (n ¼ 3), London, UK (n ¼ 1). The cryopreserved CB unit was shipped in an appropriate container cooled by liquid nitrogen in vapor phase. The unit was thawed and washed according to previously published methods. 17 After thawing, the following tests were performed on the cord blood: NC counts, quantification of CD34 þ cells by cytofluorimetric analysis, 18 clonogenic assays, cell viability and microbiological cultures. As the conditioning regimen, 32 patients received 12 Gy fractionated total body irradiation (TBI) by linear accelerator (200 cgy twice a day for six doses, given over 3 consecutive days starting on day 7), etoposide (20 mg/kg by continuous i.v. infusion over 24 h on day 4), cyclophosphamide (60 mg/kg, once daily i.v. on days 3 and 2, total dose 120 mg/kg), horse antilymphocyte globulin (Lymphoglobuline, Sangstat, Lyon, France) (600 IU/kg once daily by i.v. infusion over 16 h for 4 consecutive days starting on days 6, total four doses) and 6-methylprednisolone (6-MPr, 2 mg/kg from day 6 through day 1). Owing to technical reasons, due to either patient s age or machine availability, 10 patients were prepared with a chemotherapy-based regimen consisting of busulphan (1 mg/kg four times daily, over 4 consecutive days, starting on day 9, total dose 16 mg/kg), cyclophosphamide (50 mg/kg once a day, over 4 consecutive days, starting on day 5, total dose 200 mg/kg), horse antilymphocyte globulin (600 IU/kg once daily by i.v. infusion over 16 h for 4 consecutive days starting on day 6, total four doses), 6-MPr (2 mg/kg from day 6 through day 1) and i.v. cyclosporine (CsA, 1 mg/kg from day 7 through day 2). Graft-versus-host disease (GVHD) prophylaxis and treatment As GVHD prophylaxis, all patients received i.v. 6-MPr at a dose of 1 mg/kg daily from day 0 and i.v. CsA, administered from day 1 at the dose of 3 mg/kg, keeping drug levels between 200 and 400 ng/ml. The dose of 6-MPr was slowly tapered starting from day þ 30, while CsA was tapered 5% per week from day 50. Acute GVHD was scored by standard criteria 19 and initially treated by increasing doses of 6-MPr (3 mg/kg). Patients surviving in remission at least 100 days after transplantation with full donor chimerism were considered evaluable for chronic GVHD, graded according to the established criteria. 20 Supportive care All patients were nursed in HEPA filtered single rooms and received the same supportive care, antimicrobial prophylaxis and transfusion policy as described elsewhere. 6 Growth factors were not intentionally used in our patients. However, patients with neuthrophil counts o 10 9 /l and marrow cellularity o5% by day 25 after transplant received granulocyte colony-stimulating factor (G-CSF, Lenograstim, Aventis-Pharma) at the dose of 5 mg/kg daily. Reinfusion of autologous cryopreserved hematopoietic stem cells was given to severely neutropenic patients failing to reach a sustained neutrophil count /l after 10 days of therapy with G-CSF. Hematopoietic recovery, chimerism and engraftment Peripheral blood counts were performed daily and hematopoietic recovery was defined to occur on the first of 3 consecutive days with an absolute peripheral blood count greater than 10 9 /l for neutrophils and on the first of 7 consecutive days with /l for platelets in the absence of transfusional support. Chimerism status after transplant was evaluated by standard cytogenetics 21 or fluorescence in situ hybridization (FISH) 22 for transplant of opposite sex and by DNA polymorphism analysis, detected by PCR amplification of variable number tandem repeats (VNTR) regions. Chimerism studies were performed on BM and peripheral blood cells at days 20, 35, 60, 100, 180, 270 and 365 after transplant. 23 Engraftment with full chimerism required myeloid hematopoietic recovery with all cells in the recipient of donor origin, while the simultaneous presence of donor and recipient cells was defined as engraftment with mixed chimerism. The absence of hematopoietic recovery at 90 days after transplant and the autologous hematopoietic reconstitution were considered as engraftment failure. Statistical analysis Data were collected in an XLS database and imported into the SPSS System 7.5 for the statistical analysis. The closure date for analysis was 15 September The end points

3 were engraftment, acute and chronic GVHD, relapse, TRM, overall survival (OS), leukemia-free survival (LFS) and event-free survival (EFS). Relapse was indicated by morphological evidence of leukemia in the BM or any extramedullary site or cytogenetic recurrence of a neoplastic clone. TRM was defined as death due to nonleukemic causes. OS was calculated from transplantation to either death or last observation. LFS was defined as the time from transplantation to leukemia relapse, death or to the last observation. EFS denotes the time from transplantation to graft failure, leukemia relapse, development of lymphoproliferative disorders, death or to the last observation. The actuarial curves of OS, LFS and EFS were prepared according to the Kaplan Meier method. 24 Cumulative incidence rates calculated taking into account the appropriate competing risks 25 were used to estimate the probabilities of hematopoietic recovery, engraftment, GVHD, relapse and TRM. The log-rank test was employed to compare hazard, and two-sided P-values p5 were considered to represent statistical significance. 26 The correlation between number of total NC, CD34 þ cells and CFU-GM infused was calculated using the Spearman rank-order correlation coefficient. 27 Variables considered in univariate analysis were: age, sex and weight, diagnosis, degree of HLA matching, donor recipient sex combination, ABO compatibility, length of 1st CR, interval between start of search and transplant, cytomegalovirus (CMV) serology before transplant, disease phase at transplant, type of conditioning regimen and cell dose infused in terms of total NC, CD34 þ cells and CFU-GM. Variables with a P-value o for each end point were tested in multivariate analysis using the Cox proportional hazard regression model. 28 In all, 95% confidence intervals (CI) were reported for the main summary statistics. Results Patient characteristics The main patient characteristics are reported in Table 1. Of 42 patients, 21 were male and 21 female; median age was 12 years (range 1 42) with 10 patients older than 18 years and median bw was 40 kg (range 11 80). All patients had a high-risk leukemia: 25 ALL (intermediate n ¼ 18; advanced n ¼ 7); 13 AML (intermediate n ¼ 8; advanced n ¼ 5); three Ph-positive CML and one JMML in advanced phase. Of the seven patients transplanted in first CR, four (two AML, two ALL) had been resistant to first induction therapy and three (one AML, two ALL) presented molecular evidence of the t(4;11) cytogenetic translocation at 3 months from CR. Overall, at the time of CBTthe disease phase was intermediate in 26 cases and advanced in 16. The median time from the start of the search for a CB unit to transplant was 104 days (range ). In 40 patients, the CB unit was mismatched with the recipient for 1 (n ¼ 23) or 2 (n ¼ 17) HLA loci. Two patients showed complete HLA matching with their donors. A total of 17 patients were sex-mismatched and 27 pairs were major (n ¼ 17) or minor (n ¼ 10) ABO mismatched. All CB units were CMV negative, while 22 patients were serologically CMV positive. Table 1 Characteristics of patients No. of patients 42 Gender, M/F 21/21 Median age, years (range) 12 (1 42) Median weight, kg (range) 40 (11 80) Diagnosis and disease status ALL 25 1stCR 4 2nd CR 14 42nd CR 2 Relapse 5 AML 13 1stCR 3 2nd CR 5 42nd CR 1 Relapse 4 CML 3 2nd CP 1 AP 2 JMML 1 HLA compatibility 4/6 17 5/6 23 6/6 2 D/R sex combination M/F 8 F/F 13 F/M 9 M/M 12 ABO incompatibility Major 17 Minor 10 Patient CMV serology Negative 20 Positive 22 The median number of NC/kg of the recipient bw was (range ) at collection and (range ) at infusion. The median proportion of NC lost after thawing was 8% (range 0 44). The median number of infused CD34 þ cells 10 5 /kg of the recipient bw was 2.4 (range ) and the median number of infused CFU-GM 10 4 /kg of recipient bw was 1.6 (range ). The dose of CFU-GM 10 4 /kg of the recipient bw was o1 in 10 patients (low dose), X1 and p2 in16 (intermediate dose) and 42 in 15 (high dose). Adult patients received a median of 9 (9 2.29) CFU- GM 10 4 /kg recipient bw, which was markedly lower than the median of 2.08 (9 25.6) CFU-GM 10 4 /kg recipient bw infused in the children, although the difference was not statistically significant. Hematopoietic recovery and engraftment Three patients died at days 19, 23 and 30, without evidence of hematopoietic recovery. All the remaining 39 evaluable patients, who survived more than 30 days, recovered hematopoiesis at a median time of 29 days (range 18 85) for neutrophils and 63 days (range ) for platelets. Of these 39 patients, 12 with an absolute neutrophil count o 10 9 /l at day 25 were given G-CSF therapy and recovered hematopoiesis, which was of full donor origin in 1099

4 Four out of 39 (10%) patients experienced an autologous hematopoietic reconstitution, which occurred spontaneously in three patients (one on G-CSF) at days 27, 33 and 37, respectively and at day 74 after infusion of BM rescue in one patient who failed to engraft. Finally, cytogenetic, FISH and DNA polymorphism studies showed engraftment with full donor chimerism in 35 patients at all time points. It is noteworthy that, because of persistent aplasia and infectious complications, two patients were reinfused at days 38 and 46 after transplant with autologous cryopreserved marrow. Following this maneuver, acute-gvhd developed and engraftment of full donor origin was registered at days 85 and 73, respectively. At the time of autologous marrow infusion, the chimerism was full in both BM and PB in one patient, mixed in PB and absent in BM in the other one. For all patients, the cumulative incidence of myeloid recovery and engraftment at day 90 after transplant was 95% (95% CI: 9 6) and 90% (95% CI: 8 1), respectively. Neither the number of NC nor the CD34 þ cell dose nor the CFU-GM infused were found to significantly affect hematopoietic recovery and the three cell doses were positively correlated with each other: NC and CD34 þ cells (R ¼ 3, Po001), NC and CFU-GM (R ¼ 1, Po001), CD34 þ cells and CFU-GM (R ¼ 0, Po001). Comparing the group of 25 patients who spontaneously achieved full chimeric engraftment with the group of 17 patients who died early or recovered with autologous hematopoiesis or required G-CSF therapy, the median time to reach an absolute neutrophil count /l was 25 days (range 18 39) and 35 days (range 21 85), respectively (Po02). No substantial difference was observed between the two patient groups for either the number of total NC or number of CD34 þ cells infused; however, the former group received a median of 2.08 (range ) 10 4 CFU-GM/kg bw, which was higher, although the difference was not statistically significant (P ¼ 7), than the median of CFU-GM/kg (range ) given to the second group. At univariate analysis, no other factor was identified to influence engraftment. Graft-versus-host disease Acute GVHD was observed in 19 patients (45%) at a median time of 12 days (range 8 71) after transplant. In total, 10 (24%) patients had grade I, five (12%) grade II, one (2%) grade III and three (7%) grade IV acute GVHD. Cumulative incidence of grade II IV and III IV acute GVHD by day 100 after transplantation was 21% (CI: 2 8) and 9% (CI: 4 4), respectively. Grade III and IV acute GVHD occurred respectively in two of three patients who were rescued with autologous cryopreserved marrow following CSA discontinuation. In univariate analysis, we did not find any significant factor affecting acute GVHD. Of 29 patients who survived in CR with full donor chimerism for more than 100 days after transplant, chronic GVHD developed in nine at a median of 211 days (range ) for a cumulative incidence of 35% (CI: 1 0). Chronic GVHD was limited in three (10%) patients and extensive in six (20%). Relapse Clinical relapse was detected between 34 and 531 days (median, 161) after transplantation in nine patients: five with ALL in advanced phase, three with ALL in intermediate phase and one with AML in relapse at time of transplant, yielding a cumulative incidence of leukemia relapse of 25% (CI: ) at 4 years. Of the six patients with a chromosome marker, two died early after transplant and four are surviving in molecular CR at 5, 6, 12 and 64 months, respectively. In univariate analysis, the actuarial probability of relapse was 32% (CI 4 ) for all patients, significantly higher in CMV seropositive patients (57 vs 5%; P ¼ 1) and in patients with more advanced disease (50 vs 22%; P ¼ 2) at time of transplant. Both variables remained significant at the Cox analysis. Mortality and causes of death Nineteen patients died at a median of 170 days (range ) after transplant. The disease recurrence was the primary cause of death in eight patients and only one patient who relapsed at day 183 is alive 333 days after transplant. In 11 patients the causes of death were transplant related: acute GVHD (n ¼ 4), chronic GVHD (n ¼ 1), bacterial infections (n ¼ 2), multiorgan failure (n ¼ 2), heart failure (n ¼ 1) and EBV-related lymphoproliferative disorder (n ¼ 1). At 4 years, the cumulative incidence of TRM was 28% (CI: 7 7). In univariate analysis, the pre-transplant factors negatively affecting TRM were age older than 18 years (52 vs 21% at 2.5 years; P ¼ 3) and the dose of CFU-GM infused o /kg of the recipient bw (60 vs 12 vs 29%; P ¼ 1) (Figure 1). In multivariate analysis, the only factor associated with an increased risk of TRM was the low CFU-GM dose infused (Table 2). OS, LFS and EFS At a median follow-up of 20 months (range 4 74) after transplant, 21 (50%) patients are alive and disease-free with full donor chimerism. The 4-year probabilities of OS, LFS and EFS were 45% (CI: 7 3), 47% (CI: 0 4) and 46% (CI: 0 2), respectively. In univariate analysis, the following factors favorably affected OS: male sex (60 vs 28%, P ¼ 5), age o18 years (53 vs 30%, P ¼ 2), negative CMV serology (73 vs 27%, P ¼ 3), intermediate disease stage at time of transplant (57 vs 31%, P ¼ 4) and CFU-GM dose infused /kg bw (58 vs 56 vs 10%, P ¼ 02). The same pre-transplant factors significantly influenced LFS: male sex (64 vs 29%, P ¼ 2), age o18 years (53 vs 30%, P ¼ 5), negative CMV serology (75 vs 26%, P ¼ 2), intermediate disease stage at time of transplant (57 vs 31%, P ¼ 3) and CFU-GM dose infused /kg bw (61 vs 58 vs 10%, P ¼ 02). Finally, three factors were significantly associated with higher EFS: male sex (59 vs 31%, P ¼ 4), negative CMV serology (70 vs 27%, P ¼ 3) and CFU-GM dose infused /kg bw (61 vs 61 vs 10%, P ¼ 02). In the Cox analysis (Table 2), the most important factor affecting

5 P = 1 Low CFU-GM dose: 0 (95% CI: 3 ) Low CFU-GM dose: 9 (95% CI: 0 9) Intermediate CFU-GM dose: 2 (95% CI: 0 9) 1101 Low Intermediate High Figure 1 TRM according to the dose of CFU-GM infused 10 4 /kg of recipient bw: low o1; intermediate X1 and p2; high 42. Table 2 Multivariate analysis of factors associated to outcome parameters Factors TRM Relapse OS LFS EFS RR (95%CI) P RR (95%CI) P RR (95%CI) P RR (95%CI) P RR (95%CI) P Male sex 4 (3 8) 3 CMV negativity 0 (1 3) 3 3 (1 7) 4 Intermediate phase 1 (5 7) 3 CFU-GM 10 4 /kg High vs low 4.6 ( ) ( ) ( ) (4 1) 4 High vs intermediate patient outcomes remained the CFU-GM dose associated with CMV serology (P ¼ 04 and 3, respectively) in influencing OS (Figure 2a,b) and with patient sex (P ¼ 08 and 3, respectively) in influencing LFS (Figure 3a,b). Finally, CFU-GM dose was the only factor significantly (P ¼ 2) affecting EFS (Figure 4). Discussion Umbilical cord blood represents an hematopoietic stem cell source potentially alternative to BM for both pediatric and adult patients who are candidates to an allogeneic transplant In the unrelated CB transplant setting, several factors have been significantly associated with patient outcome. However, most of these studies are based on retrospective, multicenter analyses 2 5,7,8 and five single Center experiences, mostly including a limited number of children or adults with leukemia, have been published to date. 1,6,10 12 Herein, we report the long-term results of a prospective study on 42 consecutive patients undergoing an unrelated CBTfor high-risk leukemia in two Centers participating in the same transplant program. The results of hematopoietic recovery and engraftment in our series compare well with those reported from other unicentric and multicentric studies, in which the majority of patients had regularly received hematopoietic growth factors as part of the transplant procedures. At 90 days after transplant, all the evaluable patients recovered hematopoiesis, which was of full donor origin in 92% of them. The exclusion of methotrexate from our regimen for GVHD prophylaxis might have contributed to facilitate the hematopoietic reconstitution. However, because of prolonged aplasia, G-CSF was administered to 13 of 39 (33%) evaluable patients and 12 recovered hemopoiesis, which was of donor origin in 10. The primary use of hematopoietic growth factors in CBTremains an open question most likely requiring a prospective randomized trial in order to be answered. Patient s age and weight, leukemia type, disease status at transplant, degree of HLA mismatching and infused cell dose have been variably identified in some analyses 3 5,7,8,11,15,16 but not in others 2,10,29 to influence engraftment. In particular, the cell dose expressed as total nucleated cells 3 5,8 or CD34 þ cells 9,11 or progenitor cells 16 emerges as the main factor correlated with a successful engraftment. In our analysis we were unable to find any statistically significant factor affecting myeloid engraftment, but patients who received a higher (P ¼ 7) number of CFU-GM reconstituted full chimeric hematopoiesis significantly faster than the other patients who needed support with G-CSF or autologous BM rescue. The observation of an early autologous hematopoietic reconstitution spontaneously occurring in three patients at days

6 1102 a Low Intermediate High P = 01 High CFU-GM dose: 8 (95% CI: 8 8) Intermediate CFU-GM dose: 6 (95% CI: 4 8) Low CFU-GM dose: 0 (95% CI: 0 9) b P = 3 CMV neg.: 3 (95% CI: 3 3) CMV pos.: 7 (95% CI: 6 8) CMV pos. CMV neg Figure 2 OS according to the dose of CFU-GM infused 10 4 /kg of recipient bw: low o1; intermediate X1 and p2; high 42 (a) and CMV recipient serology (b). 27, 33 and 37 after transplant is noteworthy. The details on this finding have been reported and extensively discussed elsewhere. 23 The Minnesota group observed autologous hematopoietic recovery in four of 102 patients of its series, but this phenomenon was restricted to patients affected by nonmalignant diseases and followed a secondary graft failure after a primary engraftment. 11 The spontaneous and primary autologous hematopoietic reconstitution occurring early after CBTin leukemic patients conditioned with a highly intensive regimen has been anecdotically reported. 1,3,4 An extensive chimerism study involving a large number of patients and specifically addressed to the early hematopoietic reconstitution in recipients of an unrelated CBTshould be carried out in order to better understand the kinetics of engraftment. Furthermore, defining the chimerism status on both lymphoid and myeloid lineages is essential for CBTpatients failing engraftment and requiring autologous BM rescue. Indeed, in our series, two of three patients given autologous BM for graft failure recovered donor hematopoiesis followed by the development of fatal acute GVHD, probably triggered by the abrupt discontinuation of the immunosuppressive therapy for GVHD prevention. In our policy the autologous BM harvest remains mandatory for patients undergoing an unrelated CBT, 6,29 but its infusion as salvage therapy should be preceded by a careful chimerism study, which can drive the rationale for properly modulating immunosuppressive therapy, that is, prior administration of antilymphocyte globulin or discontinuation of CSA. To prevent GVHD, only CSA in the standard regimen combined with low dose of 6-MPr was used. The incidence of either advanced acute (II IV grade: 21%; III IV grade: 9.5%) or extensive chronic (20%) GVHD in our patients, mostly transplanted with an unrelated HLA mismatched CB unit and also including 10 adults, is considerably lower than that observed in children grafted from an unrelated HLA matched BM donor 13,30,31 and compares favorably with that reported from other studies in patients, mainly pediatric, undergoing an unrelated CBT. 3 5,7,8,11,13,29 Our policy of cord blood selection with no more than two HLA disparities with the recipient, the use of antilymphocyte globulin as part of the conditioning regimen and the modality of 6-MPr administration combined with CSA can only partially explain the low incidence of XII grade acute GVHD in our series. However, in our analysis no factor was found to significantly influence the occurrence of GVHD. According to other studies 3 5 the number of HLA disparities between unrelated cord blood and recipient were

7 a P = 02 High CFU-GM dose: 1 (95% CI: 2 0) Intermediate CFU-GM dose: 8 (95% CI: 1 5) Low CFU-GM dose: 0 (95% CI: 9) Low Intermediate High Figure 3 b P = 2 Male: 4 (95% CI: 2 6) Female: 9 (95% CI: 7 1) Female Male LFS according to the dose of CFU-GM infused 10 4 /kg of recipient bw: low o1; intermediate X1 andp2; high 42 (a) and patient sex (b). High CFU-GM dose: 1 (95% CI: 2 0) Intermediate CFU-GM dose: 1 (95% CI: 6 6) Low CFU-GM dose: 0 (95% CI: 0 9) Low Intermediate High P = 2 Figure 4 EFS according to the dose of CFU-GM infused 10 4 /kg of recipient bw: low o1; intermediate X1 andp2; high 42. not correlated with the incidence and severity of either acute or chronic GVHD. The low risk of GVHD in recipients of an unrelated, HLA mismatched CB transplant confirms in the clinical setting the observation of a reduced reactivity of cord blood Tlymphocytes against alloantigens in vitro, probably reflecting the phenotypic and functional immaturity of cord blood cells 32 and their high sensitivity to cyclosporine. 33 Furthermore, data on GVHD support the policy of omitting methotrexate from regimens of GVHD prophylaxis, therefore avoiding its potential unfavorable effect on engraftment. Relapse occurred in only nine patients of our series, six of whom were transplanted in advanced phase, for a cumulative relapse incidence of 25% at 4 years. The four evaluable patients monitored for a chromosome marker were in molecular remission at last follow-up. Despite the

8 1104 low incidence of GVHD, the graft-versus-leukemia (GVL) effect does not seem to be impaired after CBT. Indeed, case controlled studies in children comparing unrelated CBT with unmanipulated or Tcell depleted unrelated BM transplant have reported similar relapse rates. 13,14 As expected and reported by others, 5,7,11 the status of advanced disease at time of transplant significantly increased the risk of relapse. However, from our multivariate analysis CMV seropositivity of the recipient also emerged as an independent factor. Positive CMV serology was reported to be associated with relapse in children with leukemia receiving either CB from a related donor 5 or an unrelated, unmanipulated BM. 13 No difference in terms of patient characteristics was found between our CMV positive and negative patients, such that reasons underlying the correlation between CMV seropositivity and relapse risk remain unknown at present. Relapse was the primary cause of death in eight patients, while GVHD represented the main cause of mortality for five of 11 patients who died because of complications related to transplant. It is noteworthy that, despite the late hematopoietic recovery, the exposure of CBTpatients to life-threatening infections seems less than expected: only two patients in our series died of bacterial sepsis. Patient age was associated with TRM with a significantly higher number of X18 years old patients dying for causes not related to relapse. The small number of our adult patients does not allow us to identify further cutoff years of age for risk of mortality. 10 However, although an interaction between age and CFU-GM dose cannot be excluded, in Cox regression analysis CFU-GM o /kg of the recipient bw was the only significant factor negatively correlated with TRM. The cell dose expressed in terms of infused CFU-GM remained an independent variable constantly associated with the likelihood of OS, LFS and EFS. CMV positivity is known to negatively affect OS of CBTpatients, 3,11 whereas the impact of patient gender in influencing LFS is unclear at present. Indeed, a significantly better outcome was observed for male patients, whose characteristics and donor recipient combinations were not substantially different with respect to female patients. Analyzing the influence of cell dose on engraftment and other transplant-related events (autologous reconstitution, back-up autograft or allograft and death) in unrelated CBT, the graft progenitor cell content in terms of total colony-forming cells (CFC) before freezing has been identified in univariate analysis as a better outcome predictor than the number of total nucleated cells. 16 More recently, it has been steadily recommended to base the selection of cord blood units on the number of CD34 þ cells, which was found in multivariate analysis as the main factor associated with rate of engraftment, TRM and survival. 11 In our analysis, despite the significant correlation with the number of CD34 þ cells and total nucleated cells infused, the number of CFU-GM emerged as the principal variable predicting patient outcomes. The principle that more is better, referring to the quantity of CB total cells infused, should be extended to the graft quality estimated in terms of in vitro CFU-GM growth: a CB unit for transplant should be selected on the base of its proliferative potential. However, at present, the limited availability of this information in the CB banks and the absence of standardized methods for in vitro measurement of the hematopoietic growth does not allow to adopt this parameter in driving the CB search. More properly, our study provides evidence that CFU-GM cultured after thawing should be considered an important prognostic index of patient outcomes after CBT. Acknowledgements This work was supported in part by grants from AIRC, MIUR, ROMAIL, EEC for Eurocord BIOMED II QLRT , P.F.Min.Salute 2001,01,X, We thank Professor Giuseppe Cimino for molecular study on chimerism, Dr Maria Rosaria De Cuia for cytogenetics, Dr Luca Laurenti for HLA typing and Dr Ippolita Rana for medical assistance. We thank also Esterina Livesi, Mirella Gasparrini and the entire staff of the G Papa Units for providing excellent care of our patients. References 1 Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation in unrelated recipients. N Engl J Med 1996; 335: Wagner JE, Rosenthal J, Sweetman R et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft versus host disease. Blood 1996; 88: Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med 1997; 337: Rubinstein P, Carrier C, Scaradovou A et al. Outcomes among 562 recipients of placental blood transplant from unrelated donors. N Engl J Med 1998; 339: Locatelli F, Rocha V, Chastang C et al. Factors associated with outcome after cord blood transplantation in children with acute leukemia. 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