Introduction. Patients and methods

Transcription

1 CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS Transplant-related mortality and long-term graft function are significantly influenced by cell dose in patients undergoing allogeneic marrow transplantation Alida Dominietto, Teresa Lamparelli, Anna Maria Raiola, Maria Teresa Van Lint, Francesca Gualandi, Giovanni Berisso, Stefania Bregante, Carmen di Grazia, Monica Soracco, Anna Pitto, Francesco Frassoni, and Andrea Bacigalupo We have studied the impact of cell dose on short- and long-term graft function and outcome in 905 patients undergoing an unmanipulated allogeneic bone marrow transplantation (BMT) from an HLAidentical sibling (n 735), a one-antigen mismatched related donor (n 35), or a matched unrelated donor (n 135). Median number of nucleated cells infused was /kg (25th percentile /kg, 75th percentile /kg). Patients were stratified according to cells infused in 3 groups: < /kg (n 247; low dose); > /kg and < /kg (n 452; intermediate dose); and > /kg (n 206; high dose). Patients Introduction receiving high cell dose had significantly higher platelet counts on days 20, 50, 100, 180, and 365 after BMT (P <.01) and higher white blood cell counts on days 50, 100, and 180 after BMT (P <.01) as compared with other patients. The actuarial 5-year transplantrelated mortality (TRM) was 41% versus 36% versus 28% (P.01); overall survival was 45% versus 51% versus 56% (P.0008); and disease-free survival was 41% versus 42% versus 48%, respectively, (P.04) in patients receiving low, intermediate, or high cell dose. The cell dose effect was more pronounced in patients older than 30 years of age, with advanced disease, with chronic myeloid leukemia, and with alternative donors. In multivariate Cox analysis on TRM, cell dose was a significant predictor (P.002; relative risk 0.6) together with donor type (P.0001), year of transplantation (P.0001), conditioning regimen (P.02), and recipient age (P.02). In conclusion, transplantation of high marrow cell dose is associated with reduced transplant mortality and improved survival and results in improved graft function both short and long term. (Blood. 2002;100: ) 2002 by The American Society of Hematology Cell dose is recognized as an important positive predictor of outcome in allogeneic bone marrow transplantation (BMT), both in the animal model 1,2 and in human beings. 3 It is unclear whether the beneficial effect is due to a larger dose of hemopoietic progenitors or other cell subpopulations 4 : One hypothesis would be that a larger number of grafted hemopoietic progenitors would lead to faster hematologic recovery. 5 In this context, mobilized peripheral blood (PB) progenitor cell transplants have called the attention of several investigators to the kinetics of hematologic reconstitution after engraftment. Comparison of PB with bone marrow (BM) transplant recipients has revealed shorter neutropenia and thrombocytopenia in the former group: In a randomized study conducted by the European group for Blood and Marrow Transplantation (EBMT), the median day to reach a granulocyte count of /L was 12 versus 15 (P.0001) for PB versus BM grafts, respectively. 6 Similarly, the median day to reach a platelet count of /L was 15 versus 20 (P.0001). 6 Therefore, hemopoietic recovery is significantly faster in PB graft recipients. In the same study, however, transplant-related mortality (TRM) and survival were identical in the 2 groups. 6 Most retrospective and prospective studies comparing PB versus BM transplants show that, despite faster hematologic recovery, transplant-related complications and mortality are quite similar Therefore, a larger number of progenitor cells in PB grafts leads to faster hematologic recovery, with little influence on transplant mortality. In the present study we asked whether increasing the cell dose in BM grafts would influence long-term hematologic recovery and possibly TRM. We therefore studied 905 consecutive patients undergoing an allogeneic BMT and analyzed graft function, as indicated by leukocyte and platelet counts and hemoglobin levels, at different time points as well as TRM, survival, and relapse. Patients and methods Patient characteristics Between April 1976 and December 2000, 905 consecutive patients with hematologic disorders underwent an unmanipulated allogeneic BMT at our institution. The median patient age was 30 years (range, 1-66 years); the median follow-up for deceased patients was 124 days (range, days) and 2510 days (range, days) for surviving patients. The donor was an HLA-identical sibling in 735 patients, a one-antigen mismatched related donor in 35, or a matched unrelated donor in 135 patients. The median donor age was 31 years (range, 2-77 years). The median interval between diagnosis and BMT was 326 days (range, days). The diagnoses were marrow failure (n 94), acute leukemia (n 397), chronic myeloid leukemia (CML, n 298), lymphoproliferative disorders (LPDs, n 55), From the Dipartimento di Ematologia Ospedale San Martino, Genova, Italy. Submitted February 5, 2002; accepted July 10, Prepublished online as Blood First Edition Paper, August 1, 2002; DOI /blood Supported by Associazione Italiana Ricerca contro il Cancro (A.I.R.C.) Milano grant (A.B.) and Associazione Ricerca Trapianto Midollo Osseo (A.RI.T.M.O.) Genova. Reprints: Alida Dominietto, Dipartimento di Ematologia PAD 6/T, Ospedale San Martino, Genova, Italy; adominietto@smartino.ge.it. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked advertisement in accordance with 18 U.S.C. section by The American Society of Hematology 3930 BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

2 BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12 CELL DOSE, GRAFT FUNCTION, TRANSPLANT MORTALITY 3931 Table 1. Clinical data No. of patients 905 Median age, y (range) 30 (1-66) Donor-recipient sex match, no. of patients (%) Female to male 249 (28) Others 656 (72) Median donor age, y (range) 31 (2-77) Stem cell source: bone marrow, no. of patients (%) 905 (100) Donor type, no. of patients (%) HLA-identical sibling 735 (81) Mismatched related donor 35 (4) Matched unrelated donor 135 (15) Disease, no. of patients (%) SAA 94 (10) CML 298 (33) AML 234 (26) ALL 163 (18) MDS 61 (7) LPDs 55 (6) Disease state, no. of patients (%)* First CR/CP 448 (55) TBI, no. of patients (%) Yes 656 (72) No 249 (28) Interval between diagnosis and BMT, d (range) 326 ( ) Median follow-up for deceased patients, d (range) 124 (1-6533) Median follow-up for surviving patients, d (range) 2510 ( ) SAA indicates severe aplastic anemia; CML, chronic myeloid leukemia; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; MDS, myelodysplasia; LPDs, lymphoproliferative disorders; CR, complete remission; CP, chronic phase; TBI, total body irradiation; and BMT, bone marrow transplantation. *The disease state is evaluated only in leukemia patients (n 811). and myelodysplasia (MDS, n 61). Of the 811 leukemia patients, 448 patients (55%) received allogeneic BMT in first remission or in first chronic phase, and 363 (45%) were classified as advanced. Patient characteristics are summarized in Table 1. Conditioning regimen and graft-versus-host disease prophylaxis Eighty-five patients (10%) were conditioned with cyclophosphamide (CY) 200 mg/kg alone; 651 patients (72%) received CY (120 mg/kg) and total body irradiation (TBI; Gy); and 169 patients (18%) were prepared with thiotepa/cy- or busulfan/cy-containing regimens. Acute graft-versushost disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) with or without methotrexate (MTX). Intravenous CyA (1-5 mg/kg daily) was administered until day 20 and then switched to the oral route (12.5 mg/kg daily). Methotrexate was administered at doses of 10 to 15 mg/m 2 on day 1 after BMT and 8 to 10 mg/m 2 on days 3, 6, and 11 after BMT. Acute and chronic GVHD were graded according to standard criteria. 17 Stem cell harvest and infusion Bone marrow (BM) was collected using small-volume (2 ml) aspirations, 20 to 25 ml/kg of recipient body weight, 18 under general anesthesia; it was processed through small-gauge needles (20 G) to avoid losing cells in the infusion filters. In cases of major ABO incompatibility, the BM was not depleted of red cells, but patients isohemagglutinins were reduced to a titer of 1:16 or less in saline, with 1 or 2 plasma exchanges. BM was administered intravenously after completion of the conditioning regimen. Graft function Platelet and white blood cell (WBC) counts were used to evaluate graft function, as were hemoglobin levels. Statistical analysis Transplant-related mortality (TRM) is defined as death due to causes unrelated to the underlying disease: Patients relapsing are censored as surviving at the time of relapse. Disease-free survival is defined as the probability of being alive free of disease; events are death in remission, relapse, and death due to the underlying disease, whichever occurs first. The Student t and Mann-Whitney tests were used for continuous variables and the 2 test for 2 2 tables. Actuarial probabilities of transplant-related mortality and overall survival were calculated with the Kaplan-Meier method, 19 and the log-rank test was used to evaluate the differences between curves. The following factors were studied in multivariate Cox analysis for potential effect on transplant-related mortality rates: donor type, donor/recipient age, type and disease phase, cell dose, and conditioning regimen. The number-cruncher software (NCSS, version 5.0; JL Hintze, Kaysville, UT) was used to perform the analysis. Results Clinical data of patients receiving a low, intermediate, or a high cell dose The median nucleated cells infused was /kg (25th percentile /kg, 75th percentile /kg). Patients were stratified into 3 groups according to low, intermediate, or high cell dose: /kg (n 247); /kg and / kg (n 452), and /kg nucleated cells (n 206). Table 2 outlines the clinical characteristics of patients in the 3 groups. Patients in the high cell dose group were significantly older (P.0005), had significantly older donors (P.001), and comprised significantly more alternative donor transplants (P.05) and fewer patients receiving TBI (P ). Notably, all of these factors have a negative influence on TRM in univariate 2 analysis: recipient age more than 30 years (TRM 38% vs 29%, P.004), donor age 30 or more years (TRM 38% vs 28%, P.003), alternative donor transplants (TRM 44% vs 30%, P.0004), and use of non-tbi regimens (TRM 36% vs 32%, P.2). Hematopoietic recovery Platelet recovery is outlined in Figure 1 in patients stratified according to the cell dose received (low, intermediate, or high): Table 2. Clinical data according to the cell dose infused at transplantation Low dose: /kg Intermediate dose: /kg and /kg High dose: /kg Variable % of patients (n) % of patients (n) % of patients (n) P Recipient age more than 30 y 37 (83) 50 (226) 55 (124).0005 Donor age more than 31 y 39 (88) 49 (225) 55 (16).001 Advanced-phase disease* 29 (105) 47 (170) 24 (88).1 Interval between diagnosis and BMT more than 365 d 52 (112) 51 (224) 45 (98).3 Alternative donor 13 (30) 21 (94) 20 (46).05 CML diagnosis 28 (63) 35 (159) 33 (76).2 TBI regimen 83 (187) 74 (336) 58 (133) BMT indicates bone marrow transplantation; CML, chronic myeloid leukemia; and TBI, total body irradiation. *The disease phase is evaluated only in leukemia patients.

3 3932 DOMINIETTO et al BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12 Figure 1. Platelet counts. Median platelet counts after bone marrow transplantation in patients receiving low ([A] /kg; n 247), intermediate ([B] / kg and /kg; n 452), and high marrow cell dose ([C] /kg; n 206). The differences were significant between high, intermediate, and low cell dose at all intervals up to 1 year after the transplantation. *P.01. Patients in the latter group had higher platelet counts at all time points up to 1 year after transplantation, and the difference was significant at the P.01 level. Similar results were obtained when looking at WBC counts (Figure 2) on days 20, 50, and 180 after BMT (P.01). Hemoglobin levels were comparable at different time points after transplantation. Cell dose and transplant-related mortality The actuarial 5-year transplant-related mortality (TRM) was 41% versus 36% versus 28%, respectively, (P.01) in patients receiving low, intermediate, or high cell dose (Figure 3). Crude TRM within day 100 was 24% for patients receiving a low cell dose, 19% for intermediate, and 16% for a high cell dose (P.008). Crude TRM for patients surviving 100 days was 23%, 17%, and 11%, respectively, in the 3 groups (P.01). The cell dose effect on TRM was more pronounced in patients with advanced-phase disease (47% vs 33% vs 26%, P.008) as compared with patients with early-phase disease (32% vs 31% vs 21%, P.1). The same was true for older patients (age 30 years; 56% vs 35% vs 27%, P.0001) as compared with younger patients (age 30 years; 31% vs 32% vs 20%, P.1). The cell dose effect on TRM was Figure 3. Five-year TRM. Actuarial 5-year transplant-related mortality (TRM) of patients grafted with low ([A] /kg; n 247, 41%), intermediate ([B] /kg and /kg; n 452, 36%), and high cell dose ([C] /kg; n 206, 28%). P values in the log-rank test are as follows: A versus B, P.2; A versus C, P.005; and B versus C, P.05. seen in patients grafted from an alternative donor (62% vs 44% vs 33%, P.004) as well as in patients grafted from an HLAidentical sibling (37% vs 31% vs 22%, P.006). When patients were stratified according to diagnosis, the cell dose effect on TRM was most significant in patients with CML (52%, 39%, 18%, respectively, for low, intermediate, high cell dose; P.001), MDS (39%, 35%, 25%; P.006), and chronic lymphoproliferative disorders (70%, 22%, 20%; P.01) as compared with patients with acute leukemia (29%, 26%, 21%; P.3) and marrow failure (52%, 42%, 32%; P.2). Acute GVHD was scored as grade 0, I, II, III, and IV, respectively, in 92 (10%), 332 (37%), 342 (38%), 104 (11%), and 35 (4%) patients. Acute GVHD grade III to IV was 17% and 16% in patients receiving low or intermediate cell dose, and this is higher when compared with patients grafted with the high cell dose (11%; P.05). Chronic GVHD was scored as absent, limited, or extensive, respectively, in 672 (74%), 186 (21%), and 47 (5%) patients and was not different in the 3 groups (P.2). Survival and causes of death The actuarial 5-year overall survival was significantly different in patients receiving low, intermediate, or a high cell dose: 45% versus 51% versus 56% (P.0008; Figure 4). Deaths due to Figure 2. WBC counts. Median white blood cell counts after bone marrow transplantation in patients receiving low ([A] /kg; n 247), intermediate ([B] /kg and /kg; n 452), and high marrow cell dose ([C] /kg; n 206). The differences were significant between high, intermediate, and low cell dose on days 50 and 180 after BMT. *P Figure 4. Five-year overall survival. Actuarial 5-year overall survival of patients grafted with low ([A] /kg; n 247, 45%), intermediate ([B] / kg and /kg; n 452, 51%), and high cell dose ([C] /kg; n 206, 56%). P values in the log-rank test are as follows: A versus B, P.06; A versus C, P.007; and B versus C, P.2.

4 BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12 CELL DOSE, GRAFT FUNCTION, TRANSPLANT MORTALITY 3933 GVHD and infections were 28% versus 23% versus 16%, respectively, in the 3 groups, P.009. There was also a difference in the risk of being infected on day 30 after transplantation: Bacterial infections were seen in 16%, 14%, and 10% in the 3 groups (P.1); fungal infections in 32%, 11%, and 9% (P.001); and viral infections in 13%, 5%, and 4% (P.01), respectively, in the low, intermediate, and high group. Causes of TRM other than GVHD and infections were comparable (P.5). The number of deaths due to leukemia was similar in the 3 groups (17%, 14%, 14%; P.8), and this was confirmed in the Kaplan-Meier analysis. Disease-free survival The actuarial 5-year disease-free survival was significantly different in patients receiving low, intermediate, or high cell dose: 41% versus 42% versus 48% (P.02). Leukemia relapse The actuarial risk of relapse at 5 years in patients who received low, intermediate, and high cell dose was, respectively, 30% versus 33% versus 35% (P.3); for patients in first remission the actuarial risk is 19%, 23%, and 26% (P.4). Multivariate analysis The cell dose was analyzed in multivariate Cox analysis for potential effect on TRM together with 6 other clinical factors: donor type, donor age, recipient age, type and disease phase, conditioning regimen, and year of transplantation. In multivariate Cox analysis on TRM, cell dose was a significant predictor (P.002; relative risk 0.6) together with donor type (P.0001), year of transplantation (P.0001), conditioning regimen (P.02), and recipient age (P.02; Table 3). Discussion We have shown in the present study that graft function is improved both in the short and long term after transplantation of a high dose of allogeneic bone marrow cells: This results in significantly higher white blood cell counts up to 6 months after engraftment and higher platelet counts at all time intervals up to 1 year after transplantation. Although this would sound expected, we could not find this information in studies comparing different cell doses or different sources of stem cells Many reports concentrate on duration of neutropenia or thrombocytopenia and disregard peripheral blood counts beyond day 20. In the present study, instead, we could show that graft function was particularly improved beyond day 20 and even beyond day 100, suggesting a long-lasting effect of a high marrow cell dose: One-year postengraftment median platelet counts for patients receiving low, intermediate, or high marrow cell dose were /L, /L, and /L, respectively. The second finding is the strong correlation between cell dose and transplant mortality, in keeping with other studies : In multivariate analysis patients receiving a high cell dose ( / kg) had a relative risk of dying of transplant-related complications of 0.6 when compared with patients receiving a low cell dose ( /kg). This was almost entirely due to a reduction of lethal infections and GVHD: The risk of death caused by infection/ GVHD (often related) was 28%, 23%, and 16%, respectively, in patients receiving a low, intermediate, or high cell dose (P.009). Other studies have shown a positive effect of cell dose on graft-versus-host disease, 20,21,26 and the fact that we find reduced GVHD with improved graft function in patients receiving a high cell dose is in keeping with the recent demonstration of a strong correlation between severity of acute GVHD and platelet counts. 27 In the present study the actuarial 5-year TRM in patients receiving low, intermediate, or high cell dose was, respectively, 41%, 36%, and 28% (P.01). This could not be explained by a selection of patients with good prognosis, because the high cell dose group contained more patients with risk factors such as older age and alternative donor transplants, and this is confirmed in the multivariate Cox analysis. The reduction of transplant-related mortality was so strong that it produced improved overall survival. We did not find a significant difference in the leukemia relapse between the 3 groups of patients, notwithstanding a recent report on a possible favorable effect of a high cell dose. 25 However, if GVHD and TRM are reduced, it is unlikely that leukemia relapse would also be reduced. In keeping with these observations, we find that the disease-free survival was significantly reduced in patients grafted with low cell dose in comparison with intermediate and high dose (41% vs 42% vs 48%; P.02). The cell dose effect on TRM was more evident in patients at high risk for dying of transplant complications, such as patients older than 30 years of age, with advanced disease, or with CML/MDS. In patients with CML, the transplant mortality was 52%, 39%, 18%, respectively, for low, intermediate, or high cell dose, in keeping with a recent report. 28 One question that comes up concerns the cell component important in reducing transplant-related mortality. Is it stem cells, hemopoietic progenitors, lymphocytes, or other cells such as stromal or mesenchymal cells? 29 Stem cell numbers would seem important, as shown by experimental data on radioprotection 30 and clinical data in human beings on progenitor cell content and quality of hematologic reconstitution. 5,31 The question is how other cells influence stem cell function. The high lymphocyte content of Table 3. Results of multivariate Cox analysis for potential effect on TRM Variable Baseline value Compared value RR (95% CI) P Cell dose /kg /kg and /kg 0.76 ( ) /kg 0.6 ( ).002 Donor type HLA-identical sibling Alternative 2.19 ( ).0001 Year of transplantation 1987 or before 1997 or before 0.44 ( ) After ( ).0001 Conditioning regimen TBI: yes TBI: no 1.36 ( ).02 Recipient age 30 y 30 y 1.02 ( ).02 Donor age 31 y 31 y 1.01 ( ).06 Disease phase Early Advanced 1.25 ( ).06 TRM indicates transplant-related mortality; RR, relative risk; CI, confidence interval; and TBI, total body irradiation.

5 3934 DOMINIETTO et al BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12 peripheral blood grafts, producing significant acute and chronic GVHD, may counterbalance the positive effect of cell dose and produce an overall negative effect, mostly when the total cell count exceeds /kg. 32 This was also seen in a recent study on CD34 selected allografts, with a cutoff for increased TRM of /kg CD34 peripheral blood cells 33 : Thus, CD34 cells per se or their progeny, including antigen-presenting cells, 34 could have a promoting role on graft-versus-host disease and thus impair graft function. Other accessory cells, such as mesenchymal stem cells (MSCs), instead, could play a positive role on graft function, by virtue either of a suppressive effect on GVHD 35 or of a direct promoting effect on stem cells 36 : These MSCs are found in bone marrow but not in peripheral blood harvest. 4 In conclusion, this study indicates a very strong cell dose effect on graft function when using bone marrow as a stem cell source, resulting in lower transplant mortality. Data from the literature and our own institution (A.B., unpublished data, 2000) would suggest that this is not the case for peripheral blood grafts. Therefore, graft function and low transplant toxicity is not solely the result of a large CD34 cell dose and is probably influenced by other cell subpopulations. Some of these, such as mesenchymal stem cells, are being studied as candidates for the cell dose effect. At present we would recommend using a high marrow cell dose for best graft function and low transplant mortality: How to manipulate peripheral blood grafts to mimic this effect remains to be determined. Acknowledgment The great work of our nursing staff is gratefully acknowledged. References 1. Gengozian N, Makinodan T. Mortality of mice as affected by variation of the X-ray dose and number of nucleated rat bone marrow cells injected. Cancer Res. 1957;17: Uharek L, Gassmann W, Glass B, Steinmann J, Loeffler H, Mueller-Ruchholtz W. Influence of cell dose and graft-versus-host reactivity on rejection rates after allogeneic bone marrow transplantation. Blood. 1992;79: Storb R, Prentice RL, Thomas ED. Marrow transplantation for treatment of aplastic anemia: an analysis of factors associated with graft rejection. N Engl J Med. 1977;296: Bacigalupo A, Frassoni F, Van Lint MT. Bone marrow or peripheral blood as a source of stem cells for allogeneic transplants. Curr Opin Hematol. 2000;7: Bacigalupo A, Piaggio G, Podestà M, et al. Influence of marrow CFU-GM content on engraftment and survival after allogeneic bone marrow transplantation. Bone Marrow Transplant. 1995;15: Schmitz N, Beksac M, Hasenclever D, et al. Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk leukemia causes more graft-versus-host disease but results in survival identical to bone marrow transplantation [abstract]. Blood. 2000;96: Powles R, Metha J, Kulkarni S, et al. Allogeneic blood and bone marrow stem cell transplantation in hematological malignant diseases: a randomized trial. Lancet. 2000;355: Bensinger WI, Martin JP, Storer B, et al. Transplantation of bone marrow as compared with peripheral blood cells from HLA-identical relatives in patients with hematologic cancer. N Engl J Med. 2001;344: Champlin RE, Schmitz N, Horowitz MM, et al. Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. Blood. 2000;95: Bacigalupo A, Zikos P, Van Lint MT, et al. Allogeneic bone marrow or peripheral blood cell transplants in adults with hematologic malignancies: a single-center experience. Exp Hematol. 1998;26: Blaise D, Kuents M, Fortanier C, et al. Randomized trial of bone marrow versus lenograstimprimed blood cell allogeneic transplantation in patients with early stage leukemia: a report from the Société Française de Greffe de Moelle. J Clin Oncol. 2000;18: Bensinger WI, Clift R, Martin P, et al. Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation. Blood. 1996;88: Ringdén O, Remberger M, Runde V, et al. Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation. Blood. 1999;94: Pavletic ZS, Bishop MR, Tarantolo SR, et al. Hematopoietic recovery after allogeneic blood stem cell transplantation compared with bone marrow transplantation in patients with hematologic malignancies. J Clin Oncol. 1997;15: Brown RA, Adkins D, Khoury H, et al. Long-term follow-up of high-risk allogeneic peripheral blood stem cell transplant recipients: graft-versus-host disease and transplant-related mortality. J Clin Oncol. 1999;17: Guardiola P, Runde V, Bacigalupo A, et al. Retrospective comparison of bone marrow and granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells for allogeneic stem cell transplantation using HLA identical sibling donors in myelodysplastic syndromes. Blood. 2002;99: Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation. 1974;18: Bacigalupo A, Tong J, Podestà M, et al. Bone marrow harvest for marrow transplantation: effect of multiple small (2 ml) or large (20 ml) aspirates. Bone Marrow Transplant. 1992;9: Kaplan EL, Meier P. Non parametric estimation from incomplete observation. J Am Stat Assoc. 1958;53: Ringdén O, Nilsson B. Death by graft-versus-host disease associated with HLA-mismatch, high recipient age, low marrow cell dose, and splenectomy. Transplantation. 1985;40: Paulin T. Importance of bone marrow cell dose in bone marrow transplantation. Clin Transplant. 1992;6: Sierra J, Storer B, Hansen JA, et al. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA-matching and marrow cell dose. Blood. 1997;89: Davies SM, Kollman C, Anasetti C, et al. Engraftment and survival after unrelated-donor bone marrow transplantation: a report from the National Marrow Donor Program. Blood. 2000;96: Cornelissen JJ, Carston M, Kollman C, et al. Unrelated marrow transplantation for adult patients with poor-risk acute lymphoblastic leukemia: strong graft-versus leukemia effect and risk factors determining outcome. Blood. 2001;97: Barrett AJ, Ringdén O, Zhang MJ, et al. Effect of nucleated marrow cell dose on relapse and survival in identical twin bone marrow transplants for leukemia. Blood. 2000;95: Byrne JL, Stainer C, Cull G, et al. The effect of the serotherapy regimen used and the marrow cell dose received on rejection, graft-versus-host disease and outcome following unrelated donor bone marrow transplantation fore leukaemia. Bone Marrow Transplant. 2000;25: Dominietto A, Raiola AM, Van Lint MT, et al. Factors influencing hematologic recovery after allogeneic hemopoietic stem cell transplants (HSCT): graft versus host disease, donor type, cytomegalovirus infections and cell dose. Br J Haematol. 2001;112: Morariu-Zamfir R, Rocha V, Devergie A, et al. Influence of CD34 marrow cell dose on outcome of HLA-identical sibling allogeneic bone marrow transplants in patients with chronic myeloid leukaemia. Bone Marrow Transplant. 2001;27: Almeida-Porada G, Flake AW, Glimp HA, Zanjani ED. Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol. 1999;27: Pruijt JF, van Kooyk Y, Figdor CG, Willemze R, Fibbe WE. Murine hematopoietic progenitor cells with colony-forming or radioprotective capacity lack expression of the 2-integrin LFA-1. Blood. 1999;93: Reisner Y, Martelli MF. Stem cell escalation enables HLA-disparate haematopoietic transplants in leukemia patients. Immunol Today. 1999;20: Przepiorka D, Anderlini P, Ippoliti C, et al. Allogeneic blood stem cell transplantation in advanced hematologic cancers. Bone Marrow Transplant. 1997;19: Urbano-Ispizua A, Carreras E, Marin P, et al. Allogeneic transplantation of CD34 selected cells from peripheral blood from human leukocyte antigen-identical siblings: detrimental effect of a high number of donor CD34 cells? Blood. 2001;98: Herbst B, Kohler G, Mackensen A, Veelken H, Mertelsmann R, Lindemann A. CD34 peripheral blood progenitor cell and monocyte derived dendritic cells: a comparative analysis. Br J Haematol. 1997;99: Lazarus H, Curtin P, Devine S, et al. Role of mesenchymal stem cells (MSC) in allogeneic trasnplantation: early phase I clinical results [abstract]. Blood. 2000;96: Bennaceur-Griscelli A, Tourino C, Izac B, Vainchenker W, Coulombel L. Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34 CD38 low/neg human bone marrow cells. Blood. 1999;94: