Myeloid and lymphoid chimerism after T-cell-depleted bone marrow transplantation: evaluation of conditioning regimens using the polymerase chain reaction to amplify human minisatellite regions of genomic DNA

Abstract

Determining both myeloid and lymphoid chimerism after T-cell-depleted allogeneic bone marrow transplantation (BMT) could be helpful in the understanding of the biology of engraftment and could provide a rational method of assessing the ability of different conditioning regimens to promote engraftment. We prospectively investigated the role of different pretransplant conditioning regimens in 29 leukemic patients post-BMT by assessing myeloid and T-cell chimerism using a rapid and sensitive polymerase chain reaction (PCR) method. Minisatellites are hypervariable regions of DNA consisting of tandem repeats of a core nucleotide sequence, and allelic polymorphism results from differences in the number of the repeats. We used this variation to distinguish between donor and recipient cells post-BMT. Seventeen patients (9 sibling and 8 unrelated donors) received conditioning with hyperfractionated total body irradiation (TBI), thiotepa, and cyclophosphamide (Cy). Of the other 12 patients (all sibling donors), 11 received TBI plus Cy plus another agent: VP16, carboplatinum, or AZQ. One patient received TBI plus thiotepa plus VP16. All but one of the patients studied received marrow from HLA-identical donors. PCR analysis confirmed donor lymphoid engraftment within 8 days of transplant in six of six patients studied. All granulocyte DNA was of donor origin within the first 4 weeks of transplant, regardless of the conditioning regimen. The day +28 T cells were exclusively of donor origin in 14 of 17 patients who received TBI plus thiotepa plus Cy, but were mixed chimeric in 10 of 12 patients who received other conditioning regimens (P < .001). Early graft rejection was seen in one unrelated transplant recipient conditioned with TBI plus thiotepa plus Cy. Late graft failure was observed in 3 of 12 patients with mixed T-cell chimerism and in none of 16 patients with full donor chimerism at day +28. However, 5 of 16 patients who had complete T-cell chimerism at day +28 developed acute graft-versus-host disease (GVHD), whereas no patient with mixed chimerism had acute GVHD. Our results indicate that minisatellite PCR is a rapid and sensitive method for assessing chimerism post-BMT, that the donor T cells are important for consistent durable engraftment, and that TBI plus thiotepa plus Cy may be superior to the other regimens studied in inducing full donor chimerism. Larger numbers and longer follow-up are necessary to confirm these data and also to assess the relationship between complete donor T-cell chimerism and leukemia-free survival.

Characterization of Natural Suppressor Cells in Human Bone Marrow

“…Graft-versus-host disease (GVHD) is one of the main obstacles in allogeneic BM transplantation, which is now a powerful strategy for the treatment of congenital immunodeficiencies, hematological neoplasias [19,20] and aplastic anemia [21] in humans. However, the elimination of T cells from the donor BM (to prevent GVHD) is often associated with relapse of the malignancy or rejection of the BM graft [22]. In the murine model, it has been reported that NS cells prevent GVHD [23].…”

Red blood cell phenotyping is a sensitive technique for monitoring chronic myeloid leukaemia patients after T‐cell‐depleted bone marrow transplantation and after donor leucocyte infusion

“…If we consider a single normalized Bcr-Abl dose of < 10 À3 as a negative value and compare these data with RCP, the correlation between RCP and Bcr-Abl dose was 77%. relapse (Ba Èr et al, 1989(Ba Èr et al, , 1992Roy et al, 1990;Lawler et al, 1991;Mackinnon et al, 1992Mackinnon et al, , 1994Roux et al, 1992Roux et al, , 1993Gardiner et al, 1997;Bader et al, 1998). We have shown previously that mixed chimaerism, as found with cytogenetic analysis of bone marrow and RCP at 6 months after BMT, was not associated with a higher incidence of relapse.…”

Mixed chimerism is frequent after allogeneic peripheral blood stem cell transplantation with positive CD34 selection, and is not reverted by low doses of donor T‐cells add‐back

“…The occurrence of MC after allogeneic transplantation is influenced by several factors, such as the patient's characteristics (underlying disease, age) (1), transplant procedures [type of transplant and manipulation of the graft, conditioning regimen, graft‐vs.‐host disease (GVHD) prophylaxis] (2–8) and the sensitivity of the technique used for the analysis of chimerism status (9, 10). Donor T lymphocytes (11, 12) and the dose of donor CD34 + progenitor cells infused (13) are important factors in achieving the engraftment.…”