Faculty Bibliography

Human CD34+ cells have been shown to retain long-term hematopoietic engrafting potential in preclinical and clinical studies. However, recent studies of human and murine CD34- stem cells suggest that these are functionally important early progenitors. Using autologous transplantation, we investigated whether canine CD34 and CD34- marrow cells could be transduced and give rise to long-term hematopoiesis. CD34+Lin- and CD34-Lin- cell populations purified by fluorescence-activated cell sorting were separately cocultivated with retroviral vectors LN (CD34+Lin-) and LNY (CD34-Lin-), which carry the neomycin (neo) gene. After myeloablative total body irradiation (920 cGy), 3 dogs received transplants of both CD34+Lin- cells and CD34-Lin- cells and 2 dogs received only CD34-Lin- cells. Untransduced autologous marrow cells were given to ensure hematopoietic recovery. Using CFU-C assays, transduction efficiencies of CD34+Lin- cells ranged from 6% to 18% with no CFU-C formation from CD34-Lin- cells. PCR-based detection of the neo gene from WBCs was used to detect transduced cells weekly after transplantation. Additional PCR studies in 3 dogs given both CD34+Lin- and CD34-Lin- cells were performed on monocytes, granulocytes, and T cells (2 dogs, one at 7.5 months and the other at 9 months) and granulocytes (1 dog at 12 months). LN was detected up to 12 months posttransplantation in WBCs and mono-myeloid and lymphoid populations from 3 dogs receiving transplants of transduced CD34+Lin- cells. LNY was not detected at any time after transplantation in 5 dogs that received transduced CD34-Lin- cells. Whereas canine CD34+Lin- marrow cells contributed to long-term multilineage hematopoiesis, progeny of CD34-Lin- progenitor cells were not detected after transplantation in these experiments.

BACKGROUND AND OBJECTIVES/OBJECTIVE: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). EVIDENCE AND INFORMATION SOURCES/METHODS: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 18 years of age and 54% for patients with inborn errors. PERSPECTIVES/CONCLUSIONS: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.

BACKGROUND AND OBJECTIVES/OBJECTIVE:The risk of relapse after autologous bone marrow transplantation (ASCT) is high and is related to the type of malignancy and phase of the disease. The outcome for the patient who relapses after an autologous transplant is poor. Some of these patients achieve a remission with conventional chemotherapy, but it is usually short-lasting. Most of them succumb to the original disease. One further therapeutic possibility is an allogeneic transplant which would confer the potential advantage of a graft-versus-leukemia effect in addition to the lack of tumor contamination of the graft and to a high-dose intensity conditioning regimen. DESIGN AND METHODS/METHODS:We have studied the outcome of 31 patients with hematologic malignancies who underwent an allogeneic hematopoietic stem cell transplant (HSCT) after failing an autologous transplant because of relapse (n=29) or persistent aplasia (n=2). The median age at allograft was 36 years (18-55) and the interval from autograft to allograft was 21 months (3-141). The source of stem-cells was unmanipulated bone marrow (n=26) or growth-factor-mobilized peripheral blood (n=5). The donor was an HLA-identical sibling (n=7), or an alternative donor (n=24) (family mismatched n=11, or matched unrelated n=13). The conditioning regimen was cyclophosphamide and thiotepa (n=22), or cyclophosphamide and total body irradiation (n=9) Graft-versus-host disease (GvHD) prophylaxis consisted of cyclosporine (CyA) + methotrexate (MTX). RESULTS:Acute GvHD was scored as 0-I, II, or III-IV in 39%, 48%, and 13% of the patients, respectively. Sixteen patients died of transplant-related complications and one of progressive disease. With a median follow-up of 220 days (9-2104) the actuarial 2-year transplant-related mortality (TRM) was 51%, the actuarial relapse risk 37%, the actuarial survival 46%. Fifteen patients (48%) are alive in complete remission, with a median follow-up of 32 months (range 2-71). INTERPRETATION AND CONCLUSIONS/CONCLUSIONS:These data suggest that patients relapsing after an autotransplant should be screened for potential related or unrelated donors: although TRM remains high there is a definite chance of long-term disease-free survival if these patients are allografted.

One hundred nine patients with hematologic malignancies, undergoing bone marrow transplants (BMT) from unrelated donors, were randomized in 2 consecutive trials to receive or not to receive antithymocyte globulin (ATG) in the conditioning regimen, as follows: (A) 54 patients (median age, 28 years; 39% with advanced disease) were randomized to no ATG (n = 25) versus 7.5 mg/kg rabbit ATG (Thymoglobulin; Sangstat, Lyon, France) (n = 29); (B) 55 patients (median age, 31 years, 71% with advanced disease) were randomized to no ATG (n = 28) versus 15 mg/kg rabbit ATG (n = 27). Grade III-IV graft-versus-host disease (GVHD) was diagnosed in 36% versus 41% (P =.8) in the first and in 50% versus 11% (P =.001) in the second trial. Transplant-related mortality (TRM), relapse, and actuarial 3-year survival rates were comparable in both trials. In fact, despite the reduction of GVHD in the second trial, a higher risk for lethal infections (30% vs 7%; P =.02) was seen in the arm given 15 mg/kg ATG. Extensive chronic GVHD developed overall more frequently in patients given no ATG (62% vs 39%; P =.04), as confirmed by multivariate analysis (P =.03). Time to 50 x 10(9)/L platelets was comparable in the first trial (21 vs 24 days; P =.3) and delayed in the ATG arm in the second trial (23 vs 38 days; P =.02). These trials suggest that (1) 15 mg/kg ATG before BMT significantly reduces the risk for grade III-IV acute GVHD, (2) this does not translate to a reduction in TRM because of the increased risk for infections, and (3) though survival is unchanged, extensive chronic GVHD is significantly reduced in patients receiving ATG.

Serum cholinesterase (CHE) has been reported to be a significant indicator of liver function and prognosis in patients with cirrhosis. On the other hand, liver complications are frequent following allogeneic stem cell transplantation (HSCT). We therefore tested whether CHE was predictive of graft-versus-host disease and outcome in HSCT recipients. We studied 689 patients receiving a HSCT from an HLA-identical sibling (SIB) (n = 511), an alternative donor (n = 173) or a syngeneic twin (n = 5). Acute graft-versus-host disease (GVHD) was scored as 0-I, II, III-IV in 325 (47%), 279 (41%), and 85 patients (12%) respectively; 190 (28%) patients died of transplant-related complications (TRM). On day -7 the median CHE serum level was comparable in patients who either survived or died of TRM (5900 IU/l). On day 0, serum CHE levels were respectively 2310 and 2120 IU/l (P = NS) indicating the impact of the conditioning regimen. On day +7 after HSCT, the median level for surviving patients was 2598 IU/l vs 2309 IU/l for patients who subsequently died (P = 0.0002), on day +21 CHE levels were respectively 3348 vs 2528 IU/l (P < 0.00001), on day +50, 3575 vs 2358 IU/l (P < 0.00001) and on day +100 4193 vs 2729 IU/l (P < 0.00001). CHE levels on day +50 strongly correlated with aGVHD (3803 vs 3070 vs 1933 IU/l for patients with GVHD grade 0-I, II, and III-IV, respectively (P < 0.00001) and relapse (3569 for patients relapsing vs 3115 IU/l for patients not relapsing, P = 0.0006). In conclusion, (1) serum cholinesterase is a simple and reliable marker of acute GVHD and transplant-related complications; and (2) high CHE levels on day +50 predict relapse. If confirmed, the latter patients may be eligible for early reduction of immunosuppressive therapy.

We have previously shown that patients at high risk of graft-versus-host disease (GVHD) and transplant-related mortality (TRM) can be identified on day +7 following an allogeneic bone marrow transplant (BMT), based on serum bilirubin and blood urea nitrogen levels. One possible approach to reduce the risk of GVHD and TRM, is pre-emptive treatment with T cell antibodies. We report a pilot study testing the feasibility of this approach in 18 high risk patients, with a median age of 41, 83% of whom had advanced disease, undergoing an alternative donor BMT (family mismatched in five and unrelated in 13). The patients received three doses of rabbit antithymocyte globulin (ATG) (Thymoglobuline; Sangstat) 1.25 mg/kg on alternate days, starting at a median interval of 11 days (range 7-13) after BMT. Controls were 20 historical unrelated donor transplants (median age 35, 63% with advanced disease), with a high score from our original publication in 1999. The actuarial 1 year TRM of the ATG-treated patients was 40% compared to 60% for untreated controls (P = 0.06). Severe grade III-IV aGVHD developed in 27% of the ATG-treated patients, and in 55% of the controls (P = 0.08). This study indicates that early pre-emptive treatment of aGVHD in day +7 high risk patients is feasible and may lead to a reduction of aGVHD and TRM. This approach is being tested in a prospective randomized trial.

The authors have shown accelerated endothelialization on polyethylene terephthalate (PET) grafts preclotted with autologous bone marrow. Bone marrow cells have a subset of early progenitor cells that express the CD34 antigen on their surfaces. A recent in vitro study has shown that CD34(+) cells can differentiate into endothelial cells. The current study was designed to determine whether CD34(+) progenitor cells would enhance vascular graft healing in a canine model. The authors used composite grafts implanted in the dog's descending thoracic aorta (DTA) for 4 weeks. The 8-mm x 12-cm composite grafts had a 4-cm PET graft in the center and 4-cm standard ePTFE grafts at each end. The entire composite was coated with silicone rubber to make it impervious; thus, the PET segment was shielded from perigraft and pannus ingrowth. There were 5 study grafts and 5 control grafts. On the day before surgery, 120 mL bone marrow was aspirated, and CD34(+) cells were enriched using an immunomagnetic bead technique, yielding an average of 11.4 +/- 5. 3 x 10(6). During surgery, these cells were mixed with venous blood and seeded onto the PET segment of composite study grafts; the control grafts were treated with venous blood only. Hematoxylin and eosin, immunocytochemical, and AgNO(3 )staining demonstrated significant increases of surface endothelialization on the seeded grafts (92% +/- 3.4% vs 26.6% +/- 7.6%; P =.0001) with markedly increased microvessels in the neointima, graft wall, and external area compared with controls. In dogs, CD34(+) cell seeding enhances vascular graft endothelialization; this suggests practical therapeutic applications. (Blood. 2000;95:581-585)

Development of nontoxic and nonmyeloablative regimens for allogeneic hematopoietic stem-cell transplantation will decrease transplantation-related mortality caused by regimen-related toxic effects. In pursuit of this goal, a dog model of stable mixed hematopoietic chimerism was established in which leukocyte-antigen-identical litter mates are given sublethal total-body irradiation (2 Gy) before stem-cell transplantation and immunosuppression with mycophenolate mofetil and cyclosporine afterward. In the current study, we examined whether donor lymphocyte infusion (DLI) could be used as adoptive immunotherapy to convert mixed to complete donor chimerism. First, 8 mixed chimeras were given unmodified DLI between day 36 and day 414 after stem-cell transplantation. After a 10- to 47-week follow-up period, there were no significant changes in the percentage of donor engraftment. Next, we immunized the donor to the minor histocompatibility antigens (mHA) of the recipient by means of repeated skin grafting. Lymphocytes from the mHA-sensitized donor were infused between day 201 and day 651 after transplantation. All 8 recipients of mHA-sensitized DLI had conversion to greater than 98% donor chimerism within 2 to 12 weeks of the infusion. Complications from mHA-sensitized DLI included graft-versus-host disease in 2 dogs and marrow aplasia in 1. These results showed that the low-dose transplant regimen establishes immune tolerance, and mHA-sensitized DLI is required to break tolerance, thereby converting mixed to complete donor chimerism. We propose that mixed chimerism established after nonmyeloablative allogeneic stem-cell transplantation provides a platform for adoptive immunotherapy that has clinical potential in the treatment of patients with malignant diseases.

BACKGROUND:The aims of this study were to ex vivo expand canine dendritic cells and determine their phenotype and functional characteristics. METHODS:CD34+-selected cells and CD34+-depleted canine bone marrow (BM) cells were cultured in Iscove's modified medium for 14 days. Cytokines added to the cultures included human granylocyte/macrophage colony-stimulating factor 5 ng/ml, hFlt3 ligand 200 ng/ml, and human tumor necrosis factor-alpha 10 ng/ml. Cultured cells and purified subpopulations were assessed for cell surface antigen expression, morphology, and function by flow cytometric analysis, electron microscopy, and an allogeneic mixed lymphocyte reaction at day 14. RESULTS:Two main cell populations were identified, DR++(bright)/CD14- and DR+(dim)/CD14+. Ex vivo expanded CD34+-selected cells showed increased allostimulatory activity compared to both cultured CD34+-depleted cells and mononuclear cells. In contrast, ex vivo expansion from CD34+-depleted cells was unsuccessful. After sorting cells from the ex vivo expanded CD34+-selected bone marrow to enrich for DR++/CD14- cells, a 42-fold increase (median) of allostimulatory activity was observed as compared with sorted DR+/CD14+ cells (P=0.02). CONCLUSIONS:Cells with dentric cell-like phenotypes and functions can be cultured from canine CD34+-selected bone marrow cells. Future studies will address the roles of these cells in engraftment, graft versus host reactions and graft-host tolerance in a canine hematogoietic stem cell transplantaton model.

Patients with severe aplastic anemia (SAA) can be successfully treated with bone marrow transplantation (BMT) or immunosuppressive therapy (IS). The current outcome using both forms of therapy among 3,669 patients treated in Europe between 1976 and 1998 is reviewed. Significant progress has been made and the overall risk of failure is now low, with survival rates greater than 80% for both treatments. Chronic graft-versus-host disease (GvHD) remains a problem for BMT patients, and carries a high risk of lethal complications. On the other hand, IS patients are exposed to late failure due to relapse or clonal/malignant diseases. First-line BMT from identical siblings is compared with IS therapy in an intent-to-treat analysis of 1,765 patients, regardless of subsequent transplant status. The outcome of SAA patients has improved considerably over time and is influenced by patient variables such as severity of the disease and age, but also by the choice of the initial treatment.