ALBERT

Description: 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.

Description: The management of disease relapse following an allogeneic transplant is problematic. For patients with some diseases treatment with DLI alone can be successful. The use of a second allogeneic procedure has been associated with a prohibitively high TRM, especially when using myeloablative conditioning. Here we report the results of a British Society for Blood and Marrow Transplantation (BSBMT) registry retrospective multicentre analysis of 70 patients receiving a second allogeneic transplant using reduced intensity conditioning (RIC) after disease relapse following an initial allogeneic transplant. In 41 cases the first procedure was myeloablative and in 17 this was with RIC (unknown: 12). Over half of the first grafts were T cell depleted (TCD). The donor was an HLA identical sibling (48), unrelated (17) or other family member (2). In 11 cases a different donor was used at second transplant. The median age at first transplant was 39 (range: 8–69). The underlying diseases are as follows: AML 21, MDS 13, ALL 13, Lymphoma 11, CML 7, MPD 3, MM 2. For the second transplant, a variety of RIC regimens were used. Of these, 85% were fludarabine based and less than 20% include TCD. At the time of analysis, 27 patients were alive at a median follow-up of 1.7 years from second allograft (range: 0.3–7.4 years). In the cohort overall, the predicted overall survival and TRM at 2 years post second allograft were 27% and 27% respectively. Disease type was significantly associated with outcome (OS 1yr: lymphoma 82%, AML 32%, MDS 39%, ALL 41%, p=0.049). Age and disease status did not impact on OS or TRM. The median time to relapse after the first transplant was 1 year (range: 0.1–12.2). There was a significant survival advantage to those who relapsed more than a year post first allograft (2yr post second allograft: 33% vs 21%, p=0.038). In addition, patients with later relapses had a TRM of 12% at 2 years compared to 39% in those relapsing within a year (p=0.009). The incidence of acute GvHD was higher following the second procedure (43%) than the first (28%). Although aGvHD at any time did not impact on OS in the whole cohort, in those who relapsed prior to a year the presence of acute GvHD post second allograft was significantly associated with a superior outcome (p=0.001). The median time to relapse from the second transplant was 0.73 years (range = 0.1 – 1.6). The predicted relapse risk post second transplant was 52 % at 2 years. Those under the median age (p=0.029) and with acute leukaemia (1 yr post second transplant risk: AML 57%, MDS 34%, ALL 54%, lymphoma 14%; p=0.056) were at higher risk. Remission status prior to either transplant, acute or chronic GvHD post second allograft, donor type and time to first relapse did not impact significantly on relapse risk. In conclusion, compared to second myeloablative transplants, a second allograft using RIC can be performed with a low TRM in those relapsing more than one year after a first allograft, even when using UD. These patients achieved an encouraging OS of 〉 30% at 2 years. In those relapsing within a year of first transplant the development of GvHD was strongly associated with a better outcome.

Description: Determining both lymphoid chimerism and the presence of minimal residual disease after allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukemia (CML) could be helpful to the understanding of the biology of leukemic relapse in this disease. We prospectively investigated 32 patients with CML post-BMT by assessing T- cell chimerism and minimal residual disease using sensitive polymerase chain reaction (PCR) methodologies. Patients were studied between 1 and 24 months post-BMT. Thirty patients received a T-cell-depleted marrow grafts and 2 received unmanipulated marrow. All but 1 patient were conditioned with total body irradiation (TBI)+thiotepa+cyclophosphamide (Cy). The other patient received TBI+Cy as conditioning. The T cells were exclusively of donor origin in 12 of 16 patients who were tested at 1 month post-BMT, but were mixed chimeric in 11 of these patients by e or = 3 months. Once mixed T-cell chimerism was documented, no patient returned to having all donor T-cells. At a median follow-up of 12 months, minimal residual disease was present in 18 of 22 patients with mixed T-cell chimerism and in 3 of 10 patients with full donor chimerism. The actuarial molecular relapse rate at 24 months for the two groups is 91% and 33%, respectively (P 〈 .02). The finding of BCR- ABL mRNA within the first 6 months of transplant or on two consecutive assays was highly predictive of subsequent cytogenetic or hematologic relapse (P = .032 and P 〈 .02, respectively). Ten patients, 9 with mixed T-cell chimerism, have relapsed (4 clinical, 6 cytogenetic) at a median of 12 months post-BMT. These data suggest that mixed T-cell chimerism may be a marker for abrogation of graft-versus-leukemia activity that is thought to be pivotal in eradicating minimal residual disease after BMT for CML.

Description: Chronic myelogenous leukemia (CML) cells are characterized by a t(9;22) translocation, which can encode one of two chimeric P210 bcr-abl fusion proteins, comprising products of either the b2a2 or the b3a2 exon junction. The junctional sequences represent potentially immunogenic tumor-specific antigens. Despite their intracellular location, the fusion proteins might be recognized immunologically by T lymphocytes if peptides, derived from these unique sequences, are capable of presentation by the major histocompatibility complex molecules. We previously found that four peptides, 9 to 11 amino acids long, spanning the b3a2 CML breakpoint bind with high or intermediate affinity to purified HLA class I molecules A3, A11, B8, or both A3 and A11. We tested the ability of these peptides to elicit specific class I restricted cytotoxic T lymphocytes (CTLs) in vitro in HLA-matched healthy donors. In addition, a longer b3a2 CML-breakpoint-derived peptide, 25 aminoacids in length (b3a2–25), was studied for its ability to induce peptide-specific, class II-mediated, T-cell proliferation. In four of four HLA-A3 donors tested, CML-A3/A11-peptide specific CTLs were induced that killed an allogeneic HLA-A3-matched peptide pulsed leukemia cell line. In two of three HLA-A3 donors, the CML-A3/A11 peptide was able to induce killing of autologous and allogeneic HLA- matched peptide-pulsed peripheral blood mononuclear cells (PBMC). CML- A3 peptide induced peptide specific CTLs in one of the four HLA A3 donors tested. No killing was observed in two HLA-B8 and two HLA-A11 donors. PBMC from seven donors were also tested for anti b3a2–25 peptide proliferation in a thymidine incorporation assay. Specific proliferation was detected in three donors, all of the HLA-DR11 haplotype. These data represent the first evidence of a cytolytic human immune response against CML bcr-abl oncogene-derived peptides and provide a rationale for developing peptide-based vaccines for this disease.

Description: Unrelated bone marrow transplantation (BMT) is often complicated by fatal opportunistic infections. To evaluate features unique to immune reconstitution after unrelated BMT, the lymphoid phenotype, in vitro function, and life-threatening opportunistic infections after unrelated and related T-cell–depleted (TCD) BMT were analyzed longitudinally and compared. The effects of posttransplant donor leukocyte infusions to treat or prevent cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infections on immune reconstitution were also analyzed. This study demonstrates that adult recipients of TCD unrelated BMTs experience prolonged and profound deficiencies of CD3+, CD4+, and CD8+ T-cell populations when compared with pediatric recipients of unrelated BMT and adults after related BMT (P 〈 .01), that these adults have a significantly increased risk of life-threatening opportunistic infections, and that the rate of recovery of CD4 T cells correlates with the risk of developing these infections. Recovery of normal numbers of CD3+, CD8+, and CD4+ T-cell populations is similar in children after related or unrelated BMT. This study also demonstrates that adoptive immunotherapy with small numbers of unirradiated donor leukocytes can be associated with rapid restoration of CD3+, CD4+, and CD8+T-cell numbers, antigen-specific T-cell responses, and resolution of CMV- and EBV-associated disease after unrelated TCD BMT.

Description: Unrelated bone marrow transplantation (BMT) is often complicated by fatal opportunistic infections. To evaluate features unique to immune reconstitution after unrelated BMT, the lymphoid phenotype, in vitro function, and life-threatening opportunistic infections after unrelated and related T-cell–depleted (TCD) BMT were analyzed longitudinally and compared. The effects of posttransplant donor leukocyte infusions to treat or prevent cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infections on immune reconstitution were also analyzed. This study demonstrates that adult recipients of TCD unrelated BMTs experience prolonged and profound deficiencies of CD3+, CD4+, and CD8+ T-cell populations when compared with pediatric recipients of unrelated BMT and adults after related BMT (P 〈 .01), that these adults have a significantly increased risk of life-threatening opportunistic infections, and that the rate of recovery of CD4 T cells correlates with the risk of developing these infections. Recovery of normal numbers of CD3+, CD8+, and CD4+ T-cell populations is similar in children after related or unrelated BMT. This study also demonstrates that adoptive immunotherapy with small numbers of unirradiated donor leukocytes can be associated with rapid restoration of CD3+, CD4+, and CD8+T-cell numbers, antigen-specific T-cell responses, and resolution of CMV- and EBV-associated disease after unrelated TCD BMT.

Description: FDG PET scanning provides useful information that is now being integrated into treatment algorithms for patients with lymphomas. Its use in relation to allogeneic transplantation has not been assessed. We have performed a prospective study to assess the role of PET scans in directing immune manipulation (withdrawal of immune suppression and donor lymphocyte infusions (DLI)) following reduced intensity transplants (RIT) in patients with lymphoid malignancies. Methods: All patients referred to University College Hospital from May 2002 with lymphoid malignancies, suitable for RIT were invited to participate. Patients had PET and CT scans pre-RIT and at 3, 6, 9, 15 and 24 months post RIT. Patients were conditioned with fludarabine and melphalan and in vivo T cell depletion with alemtuzumab. Ciclosporin was given from day −1 to 3 months after RIT. Initial management for positive imaging was withdrawal of immune suppression. DLI was subsequently given to patients who progressed or relapsed at escalating doses of 1 x 106/kg T cells up to 1 x 108/kg. Patients with active GVHD were excluded from DLI. Results: 29 patients, median age 44 years (range 20 to 64 years) entered the study; 24 (6 Hodgkin lymphoma (HL), 2 Mantle cell (MC), 16 Non-Hodgkin Lymphoma (NHL)) are evaluable with 〉 3 months follow-up. 13 of 24 had positive PET scans before RIT, 9 had negative scans and 2 were not scanned before RIT. Of the 13 patients who had positive PET scans, there were 3 transplant related mortalities: CMV pneumonitis (n=1, HL), severe GVHD (n=2, NHL). The 10 remaining patients with positive PETs pre-RIT have a median 10.5 months follow up (range 6 to 24 months). 9 of 10 patients had a response to RIT (7CR and 2PR). Subsequently 5 of these 10 have relapsed/progressed at 6 to 9 months from RIT. Two were treated by withdrawal of immune supression. Both developed GVHD and died of progressive disease. 3 were treated with DLI. One patient (NHL) responded 3 months after 1 x106/kg, one (MC) after 1 x 107/kg and the third (NHL) showed a CR following both 1 x 107/kg and rituximab given for immune cytopenia. The remaining 5 patients (1HL, 4NHL) with positive pre-RIT PET scans remain in remission a median of 9 months (range 6 to 15 months) after RIT. One of 9 patients (NHL) with negative pre-RIT PET scans had progression of disease at 9 months, treated with escalating DLI up to 1 x 107/kg with no response. The remaining 8 patients with negative pre-RIT PET scans have remained in CR a median of 9 months (range 3 to 24 months) from RIT. One patient who did not receive pre-RIT scan died at 9 months of GVHD without evidence of disease progression. The second progressed at 12 months and is awaiting biopsy. 6 patients had nodes on CT scan of unclear significance, with negative PET scans. One of these patients has subsequently relapsed. The remaining patients continue in CR. 3 patients have had positive PET scans and received DLI 3 to 6 months before positive CT findings. This study suggests that PET imaging provides additional information beyond that available from CT scanning aiding patient management including earlier intervention (n=3) and deferring unnecessary treatment (n=6). 3/4 patients receiving DLI have responded. Positive PET scanning pre-RIT was associated with inferior outcome but does not preclude CR (6/13). Further follow-up and larger event numbers are required to establish the role of PET imaging in modulating treatment following RIT in patients with lymphoid malignancies.