Faculty Bibliography

We have analyzed 2,002 patients grafted in Europe between 1976 and 1998 from an identical twin (n = 34), from an HLA-identical sibling (n = 1,699) or from an alternative donor (n = 269), which included unrelated and family mismatched donors. The proportions of patients surviving in these three groups are, respectively, 91, 66 and 37%: major causes of failure were acute graft-versus host disease (GvHD) (11%), infection (12%), pneumonitis (4%), rejection (4%). In multivariate Cox analysis, factors predicting outcome were patient's age (p < 0.0001), donor type (p < 0.0001), interval between diagnosis and bone marrow transplantation (BMT) (p < 0.0005), year of BMT (p = 0.0005) and female donor for a male recipient (p = 0.02). Patients were then divided in two groups according to the year of BMT: up to or after 1990. The overall death rate dropped from 43 to 24% (p < 0.00001). Improvements were seen mostly for grafts from identical siblings (from 54 to 75%, p < 0.0001), and less so for alternative-donor grafts (from 28 to 35%; p = 0.07). Major changes have occurred in the BMT protocol: decreasing use of radiotherapy in the conditioning regimen (from 35 to 24%; p < 0.0001) and increasing use of cyclosporin (with or without methotrexate) for GvHD prophylaxis (from 70 to 98%; p < 0.0001). In conclusion, the outcome of allogeneic BMT for patients with severe aplastic anemia has considerably improved over the past two decades: young patients, grafted early after diagnosis from an identical sibling, have currently an over 80% chance of long-term survival. Transplants from twins are very successful as well. The risk of complications with alternative donor transplants is still high.

One hundred consecutive patients with severe aplastic anemia (SAA) received horse antilymphocyte globulin (ALG), cyclosporin A (CyA), 6-methylprednisolone (6Mpred), and granulocyte colony-stimulating factor (G-CSF) as first-line therapy. The median age was 16 years (range, 1-72 years) and median neutrophil count was 0.2 x 10(9)/L (range, 0-0.5 x 10(9)/L). Trilineage hematologic recovery (at a median interval of 96 days from treatment) was seen in 77 patients (48 complete, 29 partial) after 1 (n = 50) or more courses of ALG (n = 27). Of the 23 nonresponders, 11 patients died at a median interval of 83 days (range, 16-1132 days), 6 were considered treatment failures and underwent transplantation, and 6 were pancytopenic. Cytogenetic abnormalities were seen in 11% of patients, clonal hematologic disease in 8%, and relapse of marrow aplasia in 9%. The actuarial survival at 5 years was 87% (median follow-up 1424 days): 76% versus 98% for patients with neutrophil counts less than versus greater than 0.2 x 10(9)/L (P =.001) and 88% versus 87% for patients aged less than versus more than 16 years (P =.8). The actuarial probability of discontinuing CyA was 38%. Patients who did not achieve a white blood cell (WBC) count of 5 x 10(9)/L during G-CSF treatment have a low probability of responding (37%) and a high mortality rate (42%). This update confirms a high probability for SAA patients of becoming transfusion independent and of surviving after treatment with ALG, CyA, 6Mpred, and G-CSF, with a significant effect of neutrophil counts on outcome. Problems still remain, such as absent or incomplete responses, clonal evolution, relapse of the original disease, and cyclosporine dependence. Early transplantation, also from alternative donors, may be warranted in patients with poor WBC response to G-CSF. (Blood. 2000;95:1931-1934)

A randomized trial was carried out comparing cyclosporin A (CsA) and short-term methotrexate (MTX) versus CsA alone for graft versus host disease (GVHD) prophylaxis in patients with severe aplastic anemia (SAA) undergoing allogeneic bone marrow transplantation (BMT) from a compatible sibling. Seventy-one patients (median age, 19 years; range, 4-46 years) were randomized to receive either CsA and MTX or CsA alone for the first 3 weeks after BMT. Subsequently, both groups received CsA orally, with gradual drug reduction until discontinuation 8 to 12 months after BMT. Patients randomized in both arms had comparable characteristics and received the same preparative regimen (ie, cyclophosphamide 200 mg/kg over 4 days). The median time for neutrophil engraftment was 17 days (range, 11-31 days) and 12 days (range, 4-45 days) for patients in the CsA/MTX group and the CsA alone group, respectively (P =.01). No significant difference was observed in the probability of either grade 2, grade 3, or grade 4 acute GVHD or chronic GVHD developing in the 2 groups. The Kaplan-Meier estimates of 1-year transplantation-related mortality rates for patients given either CsA/MTX or CsA alone were 3% and 15%, respectively (P =.07). With a median follow-up of 48 months from BMT, the 5-year survival probability is 94% for patients in the CsA/MTX group and 78% for those in the CsA alone group (P =. 05). These data indicate that the use of CsA with MTX is associated with improved survival in patients with SAA who receive transplants from compatible siblings. (Blood. 2000;96:1690-1697)

Preparative regimens without total body irradiation (TBI) have been reported for alternative donor hemopoietic stem cell transplants (HSCT). Between 7 September 1994 and 7 June 1999 48 patients with advanced hematologic malignancies were conditioned with thiotepa (THIO) 15 mg/kg, cyclophosphamide (CY) 150 mg/kg and antithymocyte globulin (ATG). Donors were HLA mismatched family members (1-2 antigens) (FAM) (n = 24, median age 31 years) or HLA matched unrelated donors (UD) (n = 24, median age 34 years). GVHD prophylaxis was cyclosporine and methotrexate. Stem cell source was peripheral blood (n = 8) or bone marrow (n = 40). Hematologic recovery was seen in 42/46 (91%) evaluable patients and complete chimerism in 31/37 patients (85%). Acute GVHD grades III-IV were seen in 10/46 patients surviving 10 days (21%) and extensive chronic GVHD in 2/36 patients surviving 100 days (5%). Twenty-six patients died (54%), eight of recurrent disease (17%) and 18 of transplant-related complications (37%): main causes of TRM were GVHD (15%), infections (15%) and graft failure (4%). Twenty-two patients (46%) survive with a median follow-up of 877 days (287-1840). The actuarial 3-year survival is 49% for FAM and 42% for UD transplants. Results obtained with this regimen in unrelated grafts for advanced CML (n = 15) were not significantly different when compared to 21 concurrent UD grafts for advanced CML prepared with CY-TBI. In conclusion, the combination of THIO-CY-ATG allows engraftment of alternative donor hemopoietic stem cells. Results are similar when using unrelated matched donors or partially mismatched family donors, and not significantly different when compared to patients conditioned with CY-TBI.

Unrelated donor bone marrow transplant (UD-BMT) has become an attractive alternative source of hematopoietic cells for patients lacking a matched sibling. The aim of this paper was to report on results of the 696 UD BMTs performed in 31 Italian institutions during the first 10 years of activity of the Italian Bone Marrow Donor Registry (IBMDR). In 1989 the Italian Bone Marrow Transplant Group (GITMO) established the IBMDR to facilitate donor search and marrow procurement for patients lacking an HLA identical sibling. By end of December 1999, 260,000 HLA-A, B typed volunteer donors had been cumulatively registered and 2,620 searches had been activated for Italian patients. At least one HLA-A, B, DRB1 matched donor was found for 54% of the patients and 696 UD BMTs were performed. In 50% of cases the donor was found in the IBMDR and in 50% in 15 other Registries. The average time from search activation to transplant was 6 months for disease other than CML. For CML it was 14 months. Actuarial 12-month transplant-related mortality (TRM) was 68% in patients grafted between 1979 and 1992 and 44% for patients grafted after 1993. Twenty-eight per cent of patients developed grade III or IV acute GvHD and 24% developed extensive chronic GvHD. The rate of disease free survival at three years was 57% for patients with 1st chronic phase CML, 37% for patients with 1st or 2nd CR ALL, 31% for AML or MDS patients < or = 18 years of age and 54% for patients with inborn errors. We conclude that the IBMDR has benefited a substantial number of patients lacking a matched sibling and has facilitated the recruitment of UDs into the international donor pool. The long time required for the search is the major obstacle to the success of this programme. This suggests that early transplant and a decrease in TRM could further improve these encouraging results.

We have used dogs to study gene transfer into hematopoietic stem cells, because of the applicability of results in dogs to human transplantation and the availability of canine disease models that mimic human diseases. Previously we reported successful gene transfer into canine marrow repopulating cells, however, gene transfer efficiency was low, usually below 0.1% (Kiem et al, Hum Gene Ther 1996; 7: 89). In this study we have used CD34-enriched marrow cells to study different retroviral pseudotypes for their ability to transduce canine hematopoietic repopulating cells. Cells were divided into two equal fractions that were cocultivated for 72 h with irradiated packaging cells producing vector with different retroviral pseudotypes (GALV, amphotropic or 10A1). The vectors used contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into four dogs after a myeloablative dose of 920 cGy total body irradiation. Polymerase chain reaction (PCR) analysis of DNA from peripheral blood and marrow after transplant showed that the highest gene transfer rates (up to 10%) were obtained with the GALV-pseudotype vector. Gene transfer levels have remained stable now for more than 18 months. Southern blot analysis confirmed the high gene transfer rate. Interference studies on canine D17 cells revealed that 10A1 virus behaved like an amphotropic virus and was not able to use the GALV receptor. In summary, our results show improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched cells are transduced by cocultivation on a GALV-pseudotype packaging cell line in combination with a GALV-pseudotype vector. Furthermore, these results demonstrate that the monoclonal antibody to canine CD34 used in this study is able to enrich for hematopoietic repopulating cells.

A competitive repopulation assay in the dog was used to develop improved gene transfer protocols for hematopoietic stem cell gene therapy. Using this assay, we previously showed improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched marrow cells were cocultivated on gibbon ape leukemia virus (GALV)-based retrovirus vector-producing cells. In the present study, we have investigated the use of fibronectin fragment CH-296 and 2 growth factor combinations to further improve gene transfer efficiency. CD34-enriched marrow cells from each dog were prestimulated for 24 hours and then divided into 3 equal fractions. Two fractions were placed into flasks coated with either CH-296 or bovine serum albumin (BSA) and virus-containing medium supplemented with growth factors, and protamine sulfate was replaced 4 times over a 48-hour period. One fraction was cocultivated on irradiated PG13 (GALV-pseudotype) packaging cells for 48 hours. In 2 animals, cells of the different fractions were transduced in the presence of human FLT-3 ligand (FLT3L), canine stem cell factor (cSCF), and human megakaryocyte growth and development factor (MGDF), and in 2 other dogs, transduction was performed in the presence of FLT3L, cSCF, and canine granulocyte-colony stimulating factor (cG-CSF). The vectors used contained small sequence differences, allowing differentiation of cells genetically marked by the different vectors. After transduction, nonadherent and adherent cells from all 3 fractions were pooled and infused into lethally irradiated dogs. Polymerase chain reaction and Southern blot analysis were used to determine the persistence of the transferred vectors in the peripheral blood and marrow cells after transplantation. The highest levels of gene transfer were obtained when cells were transduced in the presence of FLT3L, cSCF, and cG-CSF (gene transfer levels of more than 10% for more than 8 months so far). Compared with the 2 animals that received cells transduced with FLT3L, cSCF, and MGDF, gene transfer levels were significantly higher when dogs received cells that were transduced in the presence of cG-CSF. Transduction on CH-296 resulted in gene transfer levels that were at least as high as transduction by cocultivation. In summary, the overall levels of gene transfer obtained with these conditions should be sufficiently high to allow stem cell gene therapy studies aimed at correcting genetic diseases in dogs as a model for human gene therapy.

BACKGROUND:Canine stem cell transplantation models have provided important preclinical information for human clinical studies. The recent cloning of cDNA for canine CD34 and the production of monoclonal antibodies that recognize canine CD34 have been the basis for the development of techniques for the large-scale enrichment of canine hematopoietic progenitor cells. In this study, we evaluated the in vivo functional properties of canine bone marrow CD34+ cells after a myeloablative conditioning regimen. METHODS:After 920 cGy total body irradiation, three dogs received infusion of autologous CD34+ selected cells from the marrow, three dogs CD34+ depleted autologous marrow cells, and two dogs received CD34+ autologous marrow cells that were immunomagnetically selected and then further purified by cell sorting. In addition, four dogs received allogeneic marrow enriched for CD34+ cells from dog leukocyte antigen-identical littermates to investigate long-term repopulating function of CD34+ cells. Chimerism studies were performed using polymerase chain reaction to detect highly polymorphic microsatellite markers. RESULTS:In three recipients of autologous marrow enriched for CD34+ cells to between 29% and 70% (1.6 x 10(6) to 3.4x10(6) CD34+ cells/kg), prompt and full hematopoietic recovery occurred, whereas in three dogs that received marrow depleted of CD34+ cells (1 x 10(7) cells/kg), no hematopoietic recovery was achieved. In two dogs that received highly purified CD34+ cells (purity: 98% and 96%, 0.79x10(6) to 0.547x 10(6) CD34+ cells/kg), delayed but full hematopoietic recovery was seen. Three of four allograft recipients of 1.75x10(6) to 6.8x10(6) CD34+ cells/kg engrafted and showed full hematopoietic recovery, whereas one dog rejected the graft. The three long-term survivors showed stable mixed hematopoietic chimerism with predominantly donor hematopoiesis. CONCLUSION/CONCLUSIONS:Transplantation of canine CD34+ cells after lethal total body irradiation provides radioprotection and gives rise to long-term hematopoietic reconstitution. Stable donor/host mixed chimerism was observed in allograft recipients most likely as a result of T-cell depletion of the grafts. Our findings suggest a future role for canine preclinical transplant studies involving in vitro manipulation of hematopoietic pro.

BACKGROUND AND OBJECTIVE/OBJECTIVE:All culture systems exploring the early (pre-CFU) hematopoietic compartment are generally complex, time-consuming and unsuitable for routine application. The aim of our study was to develop a stroma-free culture system to quantify early bone marrow (BM) myeloid progenitor cells. DESIGN AND METHODS/METHODS:Low density, progenitor cell enriched BM cells underwent a double cytotoxic treatment with CD38 and CD33 monoclonal antibodies + rabbit complement, which depleted 99% of CFU-GM and BFU-E. Then they were cultured, both in agar and in limiting-dilution liquid culture, in the presence of 5637 cell line supernatant (containing GM-CSF, G-CSF and interleukin 1 ), stem cell factor (SCF) and interleukin 3 (IL3). RESULTS:The largest number (median 14.9 on 1x10(5) cells) and size (>50,000 cells) of myelomonocytic cell clones from CD33Eth /CD38Eth progenitors was reached after 3-4 weeks of liquid culture. SCF, but not IL3, was essential for that growth. The frequency of CD33-/ CD38- progenitors grown in liquid culture was approximately three times greater than the LTC-IC frequency in the same cell suspension. An average 93% of CD33-/CD38- progenitors displayed HLA-DR antigens and 43% generated secondary CFU-GM. In the BM of 9/10 patients, previously exposed to chemotherapy, CD33-/CD38- progenitor frequency was quite low (median 0.9 on 1x10(5) cells), in spite of normal cellularity and morphology and sustained disease remission. INTERPRETATION AND CONCLUSIONS/CONCLUSIONS:CD33-/CD38- progenitors can be grown and quantified in a stroma-free culture system in a relatively short time. The test can reveal long-lasting, subclinical BM damage induced by chemotherapy and could also be valuable for estimating the amount of early myeloid progenitors for transplantation purposes.

Transplant-related mortality (TRM) following allo- geneic bone marrow transplantation (BMT) remains a major concern and early identification of patients at risk may be clinically relevant. In this study we describe a predictive score based on bilirubin and blood urea nitrogen (BUN) levels on day +7 after BMT. The patient population consisted of 309 consecutive patients who underwent BMT from sibling (n = 263) or unrelated donors (n = 46) for hematologic disorders between December 1990 and December 1996. Of 27 laboratory tests taken on day +7 after BMT, serum bilirubin (P = 0.02) and BUN (P = 0.007) were found to be independent predictors of TRM in multivariate analysis. The median levels of bilirubin (0.9 mg/dl) and of BUN (21 mg/dl) were then used as a cut-off and a score of 1 was given for values equal/greater than the median. There were 216 patients with scores 0-1 (low risk) on day +7 (bilirubin <0.9 and/or BUN <21) and 93 patients with score 2 (high risk) (bilirubin >/=0.9 and BUN >/=21): the latter had more grade III-IV acute graft-versus-host disease (P = 0.03), slower neutrophil (P = 0.02) and slower platelet engraftment (P = 0.002). The actuarial 5 year TRM is 22% for low risk vs44% for high risk patients (P = 0.0003). For HLA-identical siblings TRM is 20% vs35% (P = 0.01), for unrelated donors it is 20% vs 65% (P = 0.01). Day +7 score was highly predictive of TRM on multivariate analysis (hazard ratio 1.9, P < 0.01), after adjustment for year of transplant (P < 0.00001), unrelated vs sibling donors (P = 0.001), patient age (P = 0.01) and diagnosis (P = 0.01). These results were validated on an independent group of 82 allogeneic BMT recipients in a pediatric Unit who showed an actuarial TRM of 16% for low risk vs 46% for high risk patients (P = 0.002). This study suggests that it may be possible to identify patients with different risks of TRM on day +7 after BMT: high risk patients could be eligible for programs designed to intensify prophylaxis of post-transplant complications.