Depleted Stem Cell Transplantation Research Articles

NK Cell Activity Influences Long Term Outcome of Pediatric Leucemias After T Cell Depleted Stem Cell Transplantation,

Abstract 4141T cell depletion with magnetic microbeads can effectively reduce GvHD rates after stem cell transplantation from both mismatched related donors as well as from matched or partially matched unrelated donors. However, T cell recovery is markedly delayed after this procedure and T cell mediated antileukemic effects may be reduced. Thus, we focused on the rapidly regenerating donor derived NK cell system and addressed the question, whether its functional activity would influence the probability of relapse in a long term analysis. Temporal development of NK cell activity was monitored in 47 pediatric patients with leukemias (ALL, AML, CML, JMML) and myelodysplastic syndromes after transplantation of T cell depleted stem cells from matched unrelated (n=18) and mismatched related (haploidentical, n=29) donors with a median follow up of 7.4 years (2.1–12). 38 patients had CR1-3, 9 patients had active disease at time of transplantation. EFS and relapse rate at 5 years for the entire group were 36% and 40%, respectively (EFS and relapse rate for ALL patients in CR1-3: 50% and 36%; EFS and relapse rate for AML/MDS patients: 22% and 30%). CD34+ selection with magnetic microbeads resulted in 8×103/kg residual T cells. No posttransplant immune suppression was given. 89% of the patients had no GvHD, 9% had GvHD grade I and only 2 patients had GvHD grade II or III. NK cells recovered rapidly after transplantation (300 CD56+/μL at day 30, median), whereas T cell recovery was delayed (median: 12 CD3+/μL at day 90). NK activity was measured as specific lysis of K562 targets several times after transplantation (mean: 3 assays per patient). Four temporal patterns of lytic activity could be differentiated: consistently low, consistently high, decreasing and increasing activity. Patients with consistently high or increasing activity had significantly lower relapse probability than patients with consistently low or decreasing levels (0.18 vs 0.73 at 5 years, p<0.05). The subgroup of patients with ALL showed similar results (0.75 vs 0.14 at 5 years, p<0.05). Speed of T cell recovery had no influence. These data suggest that both achieving and maintaining a high level of NK activity may contribute to prevent relapse. Thus, this model comprises direct functional data in the form of NK cell activation levels which are likely to be influenced by the presence of different cytokines in each patient. Our observations may have some clinical implications: immunomagnetic depletion of T cells prevents GvHD and can be performed in pediatric leukemias in remission without excessive increase in relapse rates. High levels of NK activity seem to be of importance. Since NK activity could be markedly increased by in vitro stimulation with Interleukin 2 (IL-2), in vivo administration should be considered. Disclosures:No relevant conflicts of interest to declare.

Favorable Outcomes in Patients with High Donor-Derived T Cell Count Following In Vivo T Cell Depleted Reduced Intensity Allogeneic Stem Cell Transplantation

Abstract 3043In patients undergoing T cell depleted stem cell transplantation (SCT) a high rate of mixed chimerism is seen. Further, chimerism analysis does not consistently predict risks of GVHD or relapse upon withdrawal of immunosuppression (IS) in these patients. Thus, a reliable predictor for the expected evolution of mixed T cell chimerism is needed to help in clinical decision-making for withdrawal of IS and donor lymphocyte infusion (DLI). An alternative immune parameter is T cell recovery post-transplant. We combined this measure with T cell chimerism and examined the predictive value of a calculated donor-derived T cell count for clinical outcomes following SCT.Patients were enrolled in a randomized clinical trial examining two different doses of rabbit anti-thymocyte globulin (ATG 2.5 or 1.7 mg/kg/day; Thymoglobulin®, Genzyme, Cambridge, MA) given IV on day –9 through –7, followed by total body irradiation (4.5 Gray), administered in 3 doses on day –1 and 0 (NCT00709592). Donor engraftment was measured by PCR of short tandem repeat alleles in each donor and patient at 4, 8, 12, and 24 weeks following SCT on whole blood, granulocytes, and total T cells. Between 2008 and 2011, 25 patients were enrolled in this trial, and 22 patients were eligible for this analysis. Nine of the 22 patients had mixed T cell chimerism (≥5% recipient DNA) at 8 weeks post-transplant, and of these 33% (n=3) became fully donor T cell chimeric after withdrawal of IS over the ensuing weeks. One of the 9 patients had improved donor chimerism after DLI. Of the 13 patients who were full donor chimeric in T cells, one patient reverted from full donor to mixed chimerism.Donor-derived CD3+ T cell count (dd CD3) was calculated from T cell chimerism and absolute blood CD3+ T cell count. Median dd CD3+ cell count at 8 weeks following SCT was 433 ƒýL (range: 3–2464). After calculating the sum of receiver operating characteristic area under the curves (AUC), a dd CD3 of 110ƒýL was found to be the optimal cut-off value with both the highest AUC sum and the highest AUC for each measure (cumulative acute and chronic GVHD: 0.79, remission: 0.74, whole blood chimerism: 0.88), and was subsequently used to distinguish between low (<110; n=8) and high (>110; n=14) values of dd CD3. A significant correlation was found between this single dd CD3 level discriminator and higher rates of cumulative GVHD (Fishers Exact Test: p = 0.024), remission (p = 0.0524) and full donor whole blood chimerism at ≥12 weeks following SCT (p < 0.0001) in patients with high-dd CD3 cell counts. Furthermore, Bayesian analysis showed higher whole blood mixed chimerism rates (Posterior Probability = 0.99), lower GVHD rates (PP = 0.99), and lower remission rates (PP = 0.99) in the low-dd CD3 group. Both overall survival and time to relapse favored the high-dd CD3 group (vs. low-dd CD3 group). However, while the relationship was significant for relapse (p = 0.028), it was only marginally significant for overall survival (p = 0.07). When measured after withdrawal of IS, declining donor or persistent mixed T cell chimerism was observed in patients who had mixed T cell chimerism and low- dd CD3 at eight weeks post SCT (Fig 1A), whereas those with high- dd CD3 had increasing donor T cell chimerism (Fig 1B). Granulocyte chimerism too was modulated by dd CD3 in patients with mixed chimerism and early DLI arrested the decline of donor hematopoiesis in a patient with low-dd CD3. NK cell counts were significantly higher in the high-dd CD3 group than in the low-dd CD3 group at 8 weeks (p = 0.044; PP = 0.99). No significant relationships were found between dd CD3 levels and donor age, donor type, total ATG dose, and infused graft CD34+ or CD3+ cell dose. [Display omitted] In conclusion, we report the effect of an easily calculable measure of donor T cell reconstitution on clinical outcomes following SCT (particularly GVHD), stability of engraftment and disease relapse. Donor-derived CD3+ cell count may help in the decision-making regarding IS withdrawal and DLI timing in allogeneic SCT recipients. We will be confirming the optimal dd CD3 cell count parameter in larger patient cohorts with comparable disease biology for validation in regimens of varying myeloablative intensities. Disclosures:Toor:Genzyme: Research Funding. Off Label Use: ATG in stem cell transplantation.

Pre‐emptive rituximab based on viraemia and T cell reconstitution: a highly effective strategy for the prevention of Epstein–Barr virus‐associated lymphoproliferative disease following stem cell transplantation

This study investigated the efficacy of a pre-emptive strategy based on the combination of Epstein-Barr virus (EBV) viraemia and poor T cell reconstitution in preventing post-transplant lymphoproliferative disease (PTLD) following T cell depleted stem cell transplant (SCT). EBV viral load and immune reconstitution were prospectively monitored in 70 consecutive children undergoing SCT following reduced intensity conditioning with alemtuzumab. Patients who developed significant EBV viraemia (> 40 000 copies/ml blood) were treated pre-emptively with rituximab if they were within 3 months of SCT or their CD3 count was <0·3 × 10⁹ /l. Of 20/70 patients who developed significant EBV viraemia, 13 received pre-emptive rituximab. The incidence of PTLD was significantly reduced in the pre-emptive cohort compared to historical controls (1·4% vs. 21·7%, P = 0·003). This difference was more marked among viraemic patients (2·7% vs. 62·5%P < 0·0001). Patients treated with rituximab demonstrated significantly delayed B cell reconstitution at 1 year post-SCT but this was not associated with an increase in infectious mortality. In 6/6 patients >3 months post-SCT who had a CD3 count >0·3 × 10⁹ /l, reduced immunosuppression only resulted in successful resolution of EBV viraemia without PTLD. This strategy is safe and highly effective in preventing PTLD following T cell depleted SCT, and directs rituximab therapy to patients at highest risk of this complication.

Is There a Role for Autologous Stem Cell Transplantation (Auto SCT) for Patients with Acute Myelogenous Leukaemia? A Retrospective Analysis.

AbstractAbstract 1689▪ Introduction:To better counsel our patients on the role of auto SCT for patients with AML we studied consecutive individuals in CR 1 who had tissue compatible siblings and underwent allogeneic (allo) SCT with subjects who had no HLA donor and actually underwent auto SCT. Methods:Patients were in CR 1 following induction combinations containing 7 days of cyatarabine and etoposide with 3 days daunorubicin followed by similar consolidation therapy. The choice for the type of graft was based on availability of HLA identical siblings. Allogeneic donors underwent PBPC mobilisation with filgrastim and for GvHD prophylaxis grafts were exposed ex vivo to alemtuzumab 1mg/1010 mononuclear cells. Patients were prescribed cyclosporin until day 90 post transplant. Individuals lacking a donor underwent PBPC mobilization with etoposide 2 gr/m2 and harvested PBPC were cryopreserved. Patients received similar myeloablative conditioning followed by infusion of the grafts. Patients were stratified by clinical and laboratory factors as well as cytogenetic risk. The end points were TRM, DFS and OS. Results:The median presentation age for both transplant groups was 35 (14-60) years. Of the 112 consecutive patients achieving remission 37 had HLA identical siblings, but 3 relapsed and donors became unavailable in 2. Thus, autologous or allogeneic grafts were actually transplanted to 43 and 32 patients respectively. There was no significant difference in the presentation clinical features, laboratory parameters, marrow morphology or proportion of low and intermediate cytogenetic risk for both transplant options. Treatment mortality as well as relapse rate was similar (14 and 15%; 39 and 27%, respectively). At a median of 1609 and 1819 post transplant days, 56% and 63% in each group survive. In univariate analysis performance status, cytogenetic risk, morphological features of dysplasia, blast count and LDH were significant factors for survival. While for the entire group there was no difference in survival between both modalities, all patients with unfavourable cytogenetics receiving an autologous graft died of disease recurrence (3 year survival 35% vs 0%; p= 0.05). Conclusions:We conclude that patients with AML who have low or intermediate cytogenetic risk undergoing myeloablative conditioning followed by autologous or allogeneic T-cell depleted stem cell transplantation appeared to have similar outcome. However, those with unfavourable karyotype are unlikely to be cured with autologous grafts and are candidates for experimental modalities. Disclosures:No relevant conflicts of interest to declare.

Functional characterization of alloreactive T cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy

AbstractImmunotherapy with allodepleted donor T cells (ADTs) improves immunity after T cell–depleted stem cell transplantation, but infection/relapse remain problematic. To refine this approach, we characterized the expression of surface markers/cytokines on proliferating alloreactive T cells (ATs). CD25 was expressed on 83% of carboxyfluorescein diacetate succinimidyl esterdim ATs, confirming this as an excellent target for allodepletion. Seventy percent of CD25− ATs expressed CD71 (transferrin receptor), identifying this as a novel marker to target ATs persisting after CD25 depletion. Comparison of residual alloreactivity after combined CD25/71 versus CD25 immunomagnetic depletion showed enhanced depletion of alloreactivity to host with CD25/71 depletion in both secondary (2°) mixed lymphocyte reactions (P < .01) and interferon-γ enzyme-linked immunospot assays (P < .05) with no effect on third-party responses. In pentamer/interferon-γ enzyme-linked immunospot assays, antiviral responses to cytomegalovirus, Epstein-Barr virus, and adenovirus were preserved after CD25/71 allodepletion. CD25/71 ADTs can be redirected to recognize leukemic targets through lentiviral transfer of a chimeric anti-CD19ζ T-cell receptor. Finally, we have established conditions for clinically applicable CD25/71 allodepletion under European Union Good Manufacturing Practice conditions, resulting in highly effective, reproducible, and selective depletion of ATs (median residual alloreactivity to host in 2° mixed lymphocyte reaction of 0.39% vs third-party response of 62%, n = 5). This strategy enables further clinical studies of adoptive immunotherapy with larger doses of ADTs to enhance immune reconstitution after T cell-depleted stem cell transplantation.

Graft-versus-leukemia effects of transplantation and donor lymphocytes

AbstractAllogeneic transplantation of hematopoietic cells is an effective treatment of leukemia, even in advanced stages. Allogeneic lymphocytes produce a strong graft-versus-leukemia (GVL) effect, but the beneficial effect is limited by graft-versus-host disease (GVHD). Depletion of T cells abrogates GVHD and GVL effects. Delayed transfusion of donor lymphocytes into chimeras after T cell–depleted stem cell transplantation produces a GVL effect without necessarily producing GVHD. Chimerism and tolerance provide a platform for immunotherapy using donor lymphocytes. The allogeneic GVL effects vary from one disease to another, the stage of the disease, donor histocompatibility, the degree of chimerism, and additional treatment. Immunosuppressive therapy before donor lymphocyte transfusions may augment the effect as well as concomitant cytokine treatment. Possible target antigens are histocompatibility antigens and tumor-associated antigens. Immune escape of tumor cells and changes in the reactivity of T cells are to be considered. Durable responses may be the result of the elimination of leukemia stem cells or the establishment of a durable immune control on their progeny. Recently, we have learned from adoptive immunotherapy of viral diseases and HLA-haploidentical stem cell transplantation that T-cell memory may be essential for the effective treatment of leukemia and other malignancies.

The Effect of Donor Lymphocyte Infusion Dose on the Occurrence of Severe Life-Threatening Acute Graft-Versus-Host Disease Early after Reduced Intensity Conditioning T Cell Depleted Stem Cell Transplantation.

Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (SCT) is an effective form of immunotherapy for hematological malignancies. The positive effects of DLI can be offset by the development of acute Graft-versus-Host-Disease (aGvHD), resulting in significant morbidity and mortality. To reduce the incidence of severe aGvHD, DLI can be administered in escalating doses starting with a low dose at a safe time point after SCT (i.e. 6 months). To prevent early disease relapse it would be preferable to administer DLI at an earlier time point after transplantation. However, only limited data exist on the dose that can be safely and effectively used early after SCT. Furthermore, after reduced intensity conditioning (RIC) SCT, potent immune response inducing antigen presenting cells of the patient remain present for months, potentially increasing the risk for GvHD after early DLI administration. To investigate if a time dependent DLI dose - GvHD response curve exists, we retrospectively analyzed the incidence of acute GvHD in 68 patients receiving a first DLI after RIC SCT both with related (n=44) and unrelated donors (n=24) for diverse indications (AML, ALL, NHL, myeloma, CLL, CML, solid tumors). A RIC regimen was used consisting of fludarabine, busulphan and horse ATG together with donor T cell depletion using alemtuzumab in vitro added to the graft prior to infusion. This regimen induces hardly any GvHD after SCT and DLI could be administered in escalating doses to most patients, either for persisting patient chimerism in the bone marrow or for progressive disease. The study group consisted of 38 male and 30 female patients (median age 55 years, range 34–66). Out of the 68 patients 7 were not evaluable due to non-GvHD related death within 3 months after DLI. Patients transplanted with a related donor (n=40) received a median of 5 x 10E6/kg CD3+ cells (range 0.3–5) at a median time point of 6.4 months (range 3.1–13) after SCT, and those transplanted with an unrelated donor (n=21) a median of 1.5 x 10E6/kg CD3+ cells (range 0.1–2.5) at a median time point of 6.9 months (range 3.1–9.7) after SCT. aGvHD was observed in 57% of evaluable patients (34% grade 1–2 and 23% grade 3–4). Analysis showed a difference in incidence of and mortality due to aGvHD between patients who received DLI before and after 5 months after SCT. Eight of 46 patients to whom DLI was administered more than 5 months after SCT developed grade 3–4 aGvHD (17%), of which 3 patients died from causes related to the GvHD (7%). Six of 11 patients who received DLI before 5 months after SCT in moderately high doses (1–5 x 10E6/kg CD3+ cells in related patients, 0.5–1.5 x 10E6/kg CD3+ cells in unrelated patients) developed grade 3–4 aGvHD (55%), of which 5 patients died from causes related to the GvHD (45%). Based on these data the subsequent patients were given lower DLI dosages. Thus, the next 4 patients received DLI at a dose of 0.1–0.3 x 10E6/kg CD3+ cells at 3.1 to 4.4 months after SCT. They developed no or only mild (grade 1) aGvHD. One of these patients transplanted for aplastic anemia received DLI for decreasing donor bone marrow chimerism (62% to 17%), after which the graft was successfully retained (stable 64% donor chimerism). Two patients received DLI for high risk disease (biphenotypic AML and refractory aggressive NHL) and are still alive 1 year after DLI. In the patient with biphenotypic AML donor bone marrow chimerism increased after DLI from 98 to 100%, and in the patient with NHL from 97% to 99%. The remaining patient was given DLI for a rapid rejection without any response. In conclusion, in patients transplanted with a T cell depleted RIC regimen, DLI can be administered safely more than 5 months after SCT with limited risk of life-threatening grade 3-4 GvHD. Before 5 months DLI results in a high incidence of severe aGvHD and GvHD related mortality. DLI may be safely administered in very low doses (< 0.5 x 10E6/kg CD3+ cells) early after SCT, retaining a positive effect on donor bone marrow chimerism.

Donor Lymphocyte Infusion for Mixed Chimerism or Residual Disease after Reduced-Intensity T Cell Depleted Stem Cell Transplantation Results in Conversion to Full Donor Chimerism Combined with Graft Versus Tumor Responses and Limited GVHD.

We perform reduced intensity allogeneic stem cell transplantation (SCT) with HLA identical sibling and matched unrelated donors using a conditioning regimen consisting of fludarabine, busulphan and ATG combined with alemtuzumab added to the stem cell graft for extensive donor T cell depletion. With this regimen hardly any acute and chronic graft versus host disease (GVHD) are observed after SCT and mixed chimerism is induced in most patients (Barge et al, Exp Hematol 2003). In patients transplanted for malignant disease, donor lymphocytes (DLI) are administered in escalating doses from 6 to 9 months to convert mixed into complete donor chimerism and to induce graft versus tumor (GVT) responses. We report results of chimerism analysis, clinical antitumor response and GVHD after DLI. 41 patients (median age 54 years, range 37–65) transplanted with stem cells from sibling donors (29) or matched unrelated donors (12) for various malignancies received DLI with a median dose of 5 × 106 CD3+ cells/kg (range 1–5) at a median time point of 7 months after SCT (range 5.1–15). After DLI administration 35 of 38 evaluable patients (92%) developed an immune response as defined as a major decrease in patient chimerism. In 26 patients full donor chimerism was achieved, in 9 patients a low patient signal remained present (1–2%). Patient chimerism decreased from median 12% (range 1 to 86%) to 0% (range 0–2%). In most patients conversion occurred after the first DLI, in 4 patients after the second and in 1 patient after the third DLI. In 31 of 38 patients measurable disease was present before DLI. In 22 of these patients conversion from mixed to full donor chimerism coincided with clinical GVT responses. Seven patients with active myeloid disease all achieved complete remission (4 AML/MDS patients with 8–65% blasts, 2 patients with progressive CML and 1 with active CMMOL). All these patients are still in CR after a median time of 22 months. In 8 myeloma patients with measurable disease 5 complete and 1 partial response were observed. However, most responding myeloma patients subsequently developed bone or extramedullary relapses without evidence of bone marrow involvement. In 13 patients with lymphoid disease 9 responses were observed. One patient with an immunocytoma and one with T-PLL achieved CR. In 7 CLL patients 3 CR and 1 PR were observed, notably two CR occurred in patients with massive bone marrow involvement. Two of four patients with aggressive NHL showed a CR to DLI in combination with rituximab and one patient a PR to DLI. Although no regression of tumor was observed in three patients with renal cell carcinoma after DLI, disease progression was halted for several years. Grade 3–4 acute GVHD developed in 22% of patients, grade 1–2 in 39%. Limited or extensive chronic GVHD was observed in 30 and 23% of patients, respectively. GVHD responded to therapy in most patients, only in 9% of patients chronic GVHD did not resolve. Mortality due to acute and chronic GVHD was 10%. In conclusion, after T cell depleted reduced intensity SCT a state of mixed chimerism is induced in most patients, which can be converted into full donor chimerism with DLI in more than 90% of patients. Conversion to full donor chimerism is accompanied with clinical GVT responses in a high percentage of patients with acceptable GVHD.

Transplant Outcome in Acute Myeloid but Not Acute Lymphocytic Leukemia Following T Cell Depleted Allogeneic Stem Cell Transplantation May Be Favorably Influenced by Early Recovery of NK Cells.

Between 05/1994 and 07/2005, 78 patients with acute leukemia (AML=49; ALL=29) received a T cell depleted stem cell transplant from an HLA-identical sibling, with 1–2 infusions of donor T cells on days 30–100. The conditioning regimen included 12–13.5 Gy total body irradiation (TBI) and cyclophosphamide (Cy) with or without fludarabine. Cyclosporine (CSA) was given as the sole GVHD prophylaxis after transplant. The stem cell source was peripheral blood in 75 and bone marrow in 3. The T cell dose ranged between 0.2 to 2 × 105CD3+ cells/kg. AML and ALL patients were distributed equally among transplant regimen and T cell dose variations. The median CD34 cell dose was 5.4 × 106 cells/kg, (5.4 for AML, and 5.5 for ALL; p=0.50). Actuarial disease free survival (DFS), transplant related mortality (TRM) and relapse rate was 42±8, 20±6 and 40±9 % for AML and 22±10; 35±19 and 64±11% for ALL patients. The median day 30 post-transplantation lymphocyte count (LC30) for AML patients was 418/μl (31–2105) and 778/μl (265–3295) for ALL patients; p=0.063). In multivariate analysis, LC30 emerged as an independent risk factor for transplant outcome in AML but not ALL patients (see figure). In AML patients LC30 above median was associated with superior DFS, lower TRM and lower relapse. CD34 dose and LC30 were highly correlated for AML and ALL analyzed together or separately. Lymphocyte subset analysis of 20 post-transplant samples on day 30 identified a large proportion of CD56+16+ natural killer (NK) cells (median 44%, range 15–86%). Since early post-transplant NK cells are immature and unregulated by KIR-expression they may exert anti-host reactivity. The more favorable outcome for AML but not ALL patients achieving higher lymphocyte counts early post-transplant recapitulates the myeloid leukemia-restricted benefit from superior NK cell recovery observed in AML but not ALL recipients of mismatched T cell depleted stem cell transplants reported by Ruggeri et al (Blood. 1999; 94:333–9). [Display omitted]

Recipient Epstein Barr Virus (EBV) Seropositivity with IgG Antibodies Against Early Antigens (EA) Could Be a Predictive Marker for the Early Onset of Post Transplant Lymphoproliferative Disorders (PTLDs) after Allogeneic Non T Depleted Stem Cell Transplantation.

After allogeneic bone marrow (BM) or peripheral blood stem (PBSC)cells transplantation, the occurrence of PTLDs most frequently associated with EBV represent a serious clinical management problem. Identification of risk factors to allow preventive strategies could be useful. In this single center retrospective study from1999 to 2003, we analyzed the EBV virus load in the graft and the EBV serological pretransplant status from 21 recipients with a PTLD and 28 control allografted patients without PTLDs. All patients received a non T cells depleted stem cells transplantation from geno and matched unrelated identical donors for various hematological disorders. The median time from graft to he onset of PTLD was two months (range 1–8 months). All the PTLDs patients received ATG in the conditioning regimen and had an EBV real time-PCR (RT-PCR) highly positive at time of diagnostic.First, we assessed the EBV viral load in the infused cells by RT-PCR. In the PTLDs group, only 2 cases( one BM and one PBSC) contained infected EBV cells: 1,3x10e7 and 4,33x10e8 per 10e6 cells respectively. In the control group, we did not detect copies (the difference was not significant). The recipient pretransplant EBV infection status (median time: 15 days before transplant) was completed with serology anti-EA (IgA,IgG) and anti -VCA(IgA). Results are in the table 1. We found only a significant difference (p=0,00023) between the recipients with PTLDs and the control group for the early antigens positivity. Anti-EA are associated with a reactivation of EBV.In conclusion, the EBV viral load in the infused cells is not associated with the onset of PTLD. However, our findings suggest that the detection of IgG antibodies to EA antigens in the recipient pretransplant serology could be a predictive marker of the early onset of a PTLD.EBV recipient pre transplant statusanti-VCA IgGanti-VCA IgAanti-EBNA IgGanti-EA IgGanti-EA IgAPTLDs pts n=2121/21 (100%)0/21 (0%)21/21 (100%)12/21 (57%)#0/21 (0%)Control pts n=2828/28 (100%)0/28 (0%)28/28 (100%)2/28 (7%)#0/28 (0%)# p=0,00023

Fludarabine (Flu) Based Cytoreductive Regimen and T-Cell Depleted Grafts from Unrelated or Mismatched Related Donors for the Treatment of High Risk Patients with Fanconi Anemia (FA).

Between 05/98 and 06/04, 15 consecutive patients with FA received hematopoietic stem cell transplants (SCT) from alternative donors at our Center. There were 7 males and 8 females aged 5 to 24 years (median 11.5). Hematologic diagnoses included aplastic anemia (AA) (N=5), myelodysplastic syndrome (MDS) in RAEB (N=4), RAEBT (N=1) or acute myelogenous leukemia (AML) (N=5). High risk features included: Age > 20 years (n=4), prior multiple transfusions (n=11), prior androgen treatment (n=12), prior infections (n=10), or advanced MDS or AML (n=9). Eight pts had related mismatched donors transplants with respective matching at 3/6 (6/10), 4/6 (6/10), 4/6 (7/10) (n=2)), 5/6 (8/10) (n=3) and 5/6 (9/10) HLA-antigens. Seven pts had unrelated donors transplants with respective matching at 5/6 (7/10), 5/6 (8/10) (n=2), 5/6 (9/10) and 6/6 (10/10) (n=3) HLA-antigens. Cytoreduction included single dose total body irradiation (SDTBI) (450 cGy), fludarabine (Flu) (30 mg/m2 x 5) and cyclophosphamide (Cy) (10 mg/Kg x 4). Immunosuppression included rabbit anti-thymocyte globulin (Thymoglobulin) and tacrolimus for all patients. Grafts were G-CSF mobilized CD34+ and E-rosette negative (E-) peripheral blood stem cell transplants for 12 pts and soybean agglutinin negative (SBA-) and E-rosette negative marrow transplants for 3 pts. Cell doses of the grafts were 1.5 – 29.6 x 106 CD34 cells/Kg and 0 – 26 x 103 CD3 cells/Kg. As evidenced by RFLP or FISH, all 15 evaluable pts were fully engrafted and complete chimeras. Fourteen pts were evaluable for graft-versus-host disease (GvHD). GvHD of the skin and of the gut was suspected in two pts but resolved completely prior to immunosuppressive treatment. With a median follow-up of 2.5 years (range 0.2–6), 13 of 15 pts are alive and 11 of 15 are alive disease-free. There were two deaths: one pt died from sepsis/ARDS at 2 months post SCT and one pt from pneumonitis/ARDS and EBV-infection 6 months post SCT. Three pts relapsed (MDS-RAEB x 1 – AML x 2): One pt relapsed 7 months post transplant, received a 2nd transplant from the same donor following busulfan and Flu and is alive, disease-free 18 months post SCT, while the other two pts are awaiting a second SCT. In summary, this cytoreductive regimen used with T-cell depleted stem cell transplants from unrelated or HLA-mismatched related donors for the treatment of high risk patients with Fanconi anemia, results in rapid hematopoietic engraftment and lymphohematopoietic reconstitution with minimal GVHD and a high disease-free survival.

The Post-Transplant Day 30 Lymphocyte Count Strongly Correlates with Transplanted Cd34 Dose and Is Predictive for Minimal Residual Disease and Mortality in Patients with Cml Undergoing Allogeneic Stem Cell Transplants from Matched Siblings.

Between 12/1993 and 5/2004, 80 patients with chronic myeloid leukemia (CML) received a T cell depleted stem cell transplant from an HLA identical sibling, with delayed add-back of donor T cells on days 30–45 and again on day 60–100 for patients not developing ≥ grade II acute GVHD. Four transplant regimens were evaluated (1) total body irradiation (TBI) + cyclophosphamide (Cy) and standard dose (SD) cyclosporine (CSA) + bone marrow transplantation (BMT) (N=25); (2) TBI + Cy + SD-CSA + peripheral blood stem cell transplantation (PBSCT) (N=16); (3) TBI + Cy + PBSCT and low dose or no (LD/N) CSA (N=22); (4) TBI + fludarabine (Flu) + Cy and LD/N CSA (N=17). Median CD34 and CD3 doses were 4.5 x 106/kg and 0.75 x 105/kg respectively Prior to transplantation 54 patients had chronic phase (CP) and 26 had more advanced CML. Kaplan-Meier survival plots were created for overall survival, current disease free survival (DFS); and transplant-related mortality (TRM). Outcomes were analyzed using log rank tests. The outcomes for chronic versus more advanced CML were respectively 85±5% vs 36±10%, p=<0.0001 for overall survival; 13±5% vs 42±11%, p<0.01 for TRM; 76±6% vs 33.5±9% p=<0.0001 for current DFS (figure). Of patients in CP, 22/48 were negative for BCR/ABL at 3 months post transplant, increasing to 35/46 at 12 months, and 42/46 are currently disease free. Factors affecting outcome in 54 CP patients were (1) source of transplant: there was a survival benefit for the PBSCT group compared to BMT recipients (p=0.03). and (2) Day 30 lymphocyte count (LC30): there was a higher survival and lower TRM for patients with a count above the median of 300/μl: 100 vs 70±9%; p=0.003; 0 vs 26±8%, p=0.006 respectively. Similar differences were observed in the PBSCT. Molecular response (PCR for BCR/ABL negative at 3, 6, 12 and months) was also highly correlated with higher LC30 at all time points (p=<0.0001; <0.0001; 0.0004 respectively). (3) CD34 dose above the median dose of 4.5 x 106/kg in 28 CP patients was associated with 100% survival and zero TRM vs 69±9% and 27±9% for 26 patients below the median; p=0.002 and 0.003 respectively). Higher than median CD34 doses significantly correlated with molecular response at 3, 6 months. (p=<0.0001; <0.0001 respectively). Higher CD34 dose also correlated significantly with higher LC30 (p= 0.03) (figure). These results show that T cell depleted SCT for CML CP results in favorable long-term survival with low TRM. Results for more advanced disease were complicated by high relapse and TRM but showed the same improved outcome with higher CD34 doses. The results highlight a predictive importance for LC30, not only for TRM, but also for a graft versus leukemia effect. Since LC30 was highly correlated with CD34 dose, our results suggest that transplant outcome in T depleted CML allografts can be improved by transplanting more than 4.5 x 106 CD34 cells/kg. [Display omitted]

Favorable Impact of T Cell Depleted PBSCT on Transplant Outcomes Despite Delayed Donor T Cell Engraftment.

Although T cell depleted stem cell transplantation can reduce risks from GVHD, it is associated with incomplete engraftment and higher relapse rates. To mitigate these negative effects we evaluated a T cell depleted PBSCT protocol followed by delayed add-back of donor T cells. Between 2001–2004, 56 patients with hematological malignancies received a preparative regimen of total body irradiation 12Gy, fludarabine 125mg/m2, and cyclophosphamide 120mg/kg followed by a T cell depleted PBSCT from an HLA identical sibling. The T cell dose was adjusted to 2 x 104/kg in all patients. Patients not developing grade <II GVHD received 107CD3 cells/kg between days 45–100. Lineage-specific T cell and myeloid chimerism was monitored day 14, 30, 45, 60 and 90 post-transplant. Patients were aged between 11–56 (median 30y). Twenty six with CML in CP, acute leukemia in first remission, or MDS RA were designated as standard risk (SR) for transplant outcome. Thirty with more advanced or refractory disease were designated high risk (HR). Overall survival was 65±7% at a median of 1 year. Disease risk was the major factor affecting outcome with lower relapse and transplant-related mortality (TRM) and higher disease-free survival (DFS) in SR versus HR patients (3.5±3.5 vs. 42±11%, 7.5±4 vs. 17.5±8% and 85±7 vs 36±9% respectively, p< 0.0001). One patient rejected the transplant and died following two further transplant attempts. The remaining patients achieved rapid 100% donor myeloid engraftment, sustained unless relapse occurred. Donor T cell engraftment was incomplete. 0nly 12.5% of patients had full donor T cell chimerism (>95% donor T cells) on day 14, rising to 50% of patients by day 90, with persistent mixed chimerism in some patients beyond 6 months. Predictably, acute GVHD grade I–IV by d60 was significantly correlated with full T cell chimerism (p< 0.0001). There was no significant difference in day 30 lymphocyte count, CMV reactivation or transplant CD34 dose between fully or partially T cell chimeric recipients at any time point. Full T cell chimerism was significantly more frequent in HR vs SR patients (on day 90: 82 vs 24%, p= 0.002). Kaplan-Meier outcome estimates for mixed vs full T cell chimerism were therefore analyzed separately for SR and HR patients. All outcomes (relapse, TRM and DFS) were more favorable (but not significantly so) for patients who had mixed chimerism at any time point between d14–90: of 13 evaluable HR patients fully chimeric at d30 there was a 74% probability of relapse vs a 58% relapse for15 mixed chimeric patients (p=NS). No patient with SR disease relapsed. There was a non-significant DFS advantage for d30 mixed vs full chimeric patients: 95% vs 85%, and 40% vs 20% for SR and HR patients respectively. These results indicate 1) full T cell chimerism is more frequent in HR patients (who have received large cumulative doses of prior chemotherapy) 2) T cell depleted PBSCT have a high frequency of prolonged mixed T cell chimerism. This does not predispose to late graft rejection, is favorable for reducing GVHD and does not negatively affect CMV reactivation, relapse, TRM and DFS.

Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell–depleted stem cell transplantation

We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence.

Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell–depleted stem cell transplantation

We evaluated the efficacy, toxicity, and outcome of preemptive ganciclovir (GCV) therapy in 80 cytomegalovirus (CMV)-seropositive patients allografted between 1991 and 1996 and compared their outcome to 35 seronegative patients allografted during the same period. Both cohorts were comparable with respect to diagnosis and distribution of high- versus standard-risk patients. All patients received a stem cell graft from an HLA-identical sibling donor, and grafts were partially depleted of T cells in 109 patients. Patients were monitored for CMV antigenemia by leukocyte expression of the CMV-pp65 antigen. Fifty-two periods of CMV reactivation occurring in 30 patients were treated preemptively with GCV. A favorable response was observed in 48 of 50 periods, and only 2 patients developed CMV disease: 1 with esophagitis and 1 with pneumonia. Ten of 30 treated patients developed GCV-related neutropenia (less than 0.5 × 109/L), which was associated with a high bilirubin at the start of GCV therapy. Overall survival at 5 years was 64% in the CMV-seronegative cohort and 40% in the CMV-seropositive cohort (P = .01). Increased treatment-related mortality accounted for inferior survival. CMV seropositivity proved an independent risk factor for developing acute graft-versus-host disease, and acute graft-versus-host disease predicted for higher treatment-related mortality and worse overall survival in a time-dependent analysis. We conclude that, although CMV disease can effectively be prevented by preemptive GCV therapy, CMV seropositivity remains a strong adverse risk factor for survival following partial T-cell–depleted allogeneic stem cell transplantation.

Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell–depleted stem cell transplantation

AbstractWe evaluated the efficacy, toxicity, and outcome of preemptive ganciclovir (GCV) therapy in 80 cytomegalovirus (CMV)-seropositive patients allografted between 1991 and 1996 and compared their outcome to 35 seronegative patients allografted during the same period. Both cohorts were comparable with respect to diagnosis and distribution of high- versus standard-risk patients. All patients received a stem cell graft from an HLA-identical sibling donor, and grafts were partially depleted of T cells in 109 patients. Patients were monitored for CMV antigenemia by leukocyte expression of the CMV-pp65 antigen. Fifty-two periods of CMV reactivation occurring in 30 patients were treated preemptively with GCV. A favorable response was observed in 48 of 50 periods, and only 2 patients developed CMV disease: 1 with esophagitis and 1 with pneumonia. Ten of 30 treated patients developed GCV-related neutropenia (less than 0.5 × 109/L), which was associated with a high bilirubin at the start of GCV therapy. Overall survival at 5 years was 64% in the CMV-seronegative cohort and 40% in the CMV-seropositive cohort (P = .01). Increased treatment-related mortality accounted for inferior survival. CMV seropositivity proved an independent risk factor for developing acute graft-versus-host disease, and acute graft-versus-host disease predicted for higher treatment-related mortality and worse overall survival in a time-dependent analysis. We conclude that, although CMV disease can effectively be prevented by preemptive GCV therapy, CMV seropositivity remains a strong adverse risk factor for survival following partial T-cell–depleted allogeneic stem cell transplantation.