Unmanipulated Grafts Research Articles

Regulatory and Naïve T Cells in Unmanipulated Donor Grafts Are Not Associated with Acute Graft Vs Host Disease in Matched Sibling Transplants for AML

Acute graft-versus-host disease (aGVHD) occurs in approximately 20–50% of matched-related stem cell transplants (HCT). The presence of increased frequencies of CD4+CD25+ regulatory T cells (Treg) in murine donor grafts has been shown to ameliorate aGVHD. In similar models, several groups have demonstrated that the transfer of equivalent numbers of naïve T cells (vs. various memory subsets) confers a relatively greater risk for aGVHD. To test the hypothesis that natural variations in donor graft naïve and Treg content would be associated with the incidence of aGVHD in graft recipients, we conducted a detailed analysis of donor graft T cells within unmanipulated grafts of 70 SCT recipients transplanted for AML/MDS following the administration of a uniform fludarabine/busulfan conditioning regimen. Median follow up time was 38 months. 19% (n=13) of recipients experienced grades II–IV aGVHD (n=3 grades III–IV). Cryopreserved donor grafts samples were analyzed using 9-color flow cytometry (with a panel including a viability dye and MAbs recognizing CD4, CD8, CD25, CD127, CD45RA, CD27, Foxp3, and Ki-67). Tregs were defined as being CD4+CD25+CD127loFoxp3+. CD45RA and CD27 were used to measure naïve (CD45RA+CD27+) and memory conventional CD4+ and CD8+ T cells. In addition, the proliferating fraction of all cells was defined by Ki-67 nuclear-antigen expression. We conducted analyses based on donor graft T cells assessed both continuously and by quartiles, with similar results. No significant associations were found between aGVHD incidence and total Tregs (HR 1.01, 95%CI 0.9–1.2, p 0.8), nor with proliferating Tregs (HR 1.2, 95%CI 0.2–5.9, p 0.8). Since murine models typically fix the dose of Tregs vs. conventional T cells (Tcon), we also analyzed GVHD risk by donor graft Treg:Tcon ratio, finding no association (HR 1.03, 95%CI 0.8–2.1, p 0.3). Although patients in the highest Treg:Tcon quartile received Treg doses known in vitro to induce suppression (>1:21.5), there was no reduction in aGVHD (HR 1.5 Quartile 4 vs others, 95%CI 0.5–4.9, p 0.5). Additionally, no significant association with aGVHD was found with total naïve T cells (HR 1, 95%CI 0.9–1.0, p 0.9), naïve CD4+ (HR 1, 95%CI 0.9–1.03, p 0.6), or naïve CD8+ cells (HR 0.98, 95%CI 0.9–1.03, p 0.6). Because naïve:memory cell ratios were also fixed in murine models demonstrating the importance of naïve T cells in GVHD, we also assessed naïve:memory T cell ratios within donor grafts with respect to recipient aGVHD. No reduction in aGVHD was observed considering naïve:memory ratios analyzed continuously (HR 1.1, 95%CI 0.3–4.2, p 0.8) or in the quartile receiving the lowest naïve:memory ratio (HR 0.5 Quartile 1 vs others, 95%CI 0.1–2.2, p 0.3). Our results demonstrate that donor graft Treg and naïve T cell content, assessed in the context of unmanipulated PBSC transplantation in matched siblings, does not predict recipient aGVHD incidence. These data suggest that the therapeutic efficacy of Treg enrichment and/ or naïve T cell depletion may require grafts to be manipulated to supraphysiologic levels for maximal therapeutic benefit.

Possible Epigenetic Regulation of Functionally Opposing Groups of Genes Involved in Ex Vivo Expansion of Cord Blood Stem Cells

Recently, we have demonstrated that modifications of chromatin involving DNA methylation and histone acetylation events can change the fate of hematopoietic stem cells (HSC), likely as a result of altered gene expression patterns (Araki H et al. Blood 2007). In our current studies we have examined the epigenetic regulation of genes induced during the expansion of HSC by chromatin modifying agents [5aza-2-deoxycytidine (5azaD), trichostatin A (TSA)]. The expression of the polycomb repressing complex gene, enhancer of zeste homolog 2 (Ezh2) has been implicated in histone methylation and deacetylation. We observed a significant increase in the transcript levels of Ezh2 in addition to self-renewal genes HoxB4, Bmi1 and GATA-2 in 5azaD/TSA treated expanded human cord blood (CB) cells. Ezh2 overexpression has been shown to prevent HSC exhaustion in a murine model by chromatin stabilization. CB CD34+ cells expanded in 5azaD/TSA resulted in retention of relatively higher transcript levels of DNA methyltransferase (DNMT), DNMT1 and lower levels of DNMT3a and DNMT3b in CD34+CD90+ cells, the most primitive subpopulation, in contrast to the relatively mature CD34−CD90− cells. The retention of higher DNMT1, the maintenance methyltransferase, in the most primitive CD34+CD90+ cells may favor the chromosomal stability of expanded grafts. Cytogenetic analysis and DNA ploidy of expanded grafts were normal and the telomere length of expanded CB grafts did not reveal any significant alteration of their lengths in comparison to unmanipulated primary CB grafts. In order to explore the roles of epigenetic modifications on the differentiation of HSC during ex vivo expansion, we have also examined the transcript levels of genes associated with differentiation in 5azaD/TSA expanded cells in comparison to cells expanded in cytokines alone. Interestingly, the transcript levels of differentiation associated genes Pu.1 and GATA-1 decreased following 5azaD/TSA treatment. The lower transcript levels of differentiation inducing genes (Pu.1, GATA-1) and higher levels of self-renewing genes (Ezh2, HoxB4) in 5azaD/TSA expanded cells suggest a possible interaction among these functionally opposing groups of genes which are likely regulated by epigenetic mechanisms. By using a chromatin immunoprecipitation (ChIP) assay we observed significantly increased levels of histone H4 acetylation on the promoter sites of Hox-B4, Bmi-1, and GATA-2 and decreased levels of acetylation on the promoter sites of genes involved in differentiation (Pu.1, GATA-1) in 5azaD/TSA treated CB cells. The degree of histone H4 acetylation of these promoters sites directly correlated with the transcript levels of these genes as evidenced by real time quantitative PCR. Futhermore, silencing of HoxB4 by using short hairpin RNA (shRNA) during 5azaD/TSA mediated expansion resulted in about a 50% reduction in the expansion of CB cells in contrast to cells containing non-silencing control shRNA, indicating HoxB4 is required for HSC expansion. Understanding how the possible interactions between functionally opposing groups of genes regulating the expansion and differentiation of HSC is mediated by epigenetic mechanisms may further facilitate the optimization of ex vivo expansion strategies of CB grafts for therapeutic use.

Allo-SCT in children with high-risk leukemia using unmanipulated grafts from alternative donors

Allogeneic HSCT is a curative treatment for high-risk leukemia. In Europe, approximately 15% of children have an HLA-matched sibling, but in 65-70% HLA allele-matched (9-10/10) unrelated donors (UD) can be identified. Transplantation using an HLA partially mismatched donor, unrelated cord blood or haploidentical family donor with graft manipulation is then considered with preference on the basis of local experience and/or availability. Here we evaluate the outcomes of 87 consecutive patients with leukemia transplanted with unmanipulated graft from matched or partially mismatched UD or cord blood (CB) at our institution between January 2001 and December 2007. Within the median follow-up of 30 months, the acute GVHD grade II was diagnosed in 70.9% patients; grades III-IV only in 4.6%. The overall incidence of chronic GVHD was 43.3% (extensive in 34.9%). The probability of 3-year EFS was 59.5% and that of 3-year overall survival was 66.9%. TRM at day +100 was 4.5%, and overall it was 13.8%. Fourteen patients (16.1%) died as a consequence of post-transplant leukemia relapse. We conclude that the prognosis of patients transplanted for leukemia using unmanipulated grafts from HLA-matched or partially mismatched UD or CB is comparable and satisfactory. TRM and relapse rate are lower than in the earlier period.

Radioimmunotherapy with [188Re]-labelled anti-CD66 antibody in the conditioning for allogeneic stem cell transplantation for high-risk acute myeloid leukemia

Between July 2000 and June 2003 a total of 21 patients with high-risk acute myeloid leukemia (AML; n = 14), AML after myelodysplastic syndrome (MDS; n = 6) or advanced MDS (n = 1) were treated with an 188-Re labelled anti-CD66 antibody in the conditioning regimen for allogeneic stem cell transplantation. Radioimmunotherapy (RIT) was followed by standard full-dose conditioning with busulfan and high-dose cyclophosphamide in 11 patients and reduced intensity conditioning regimen in 10 patients. All patients received an unmanipulated allogeneic graft from alternative donors (n = 15) or a HLA-identical familiy donor (n = 6). With a median follow up of 42 months (23-60) disease free survival for all patients was 43%. Nine patients are still alive and in ongoing complete hematological remission. The treatment related mortality was 28.6% (n = 6) and an equal number of patients died of relapsing disease within 30-385 days after transplantation. Late organ toxicity, monitored for more than 1 year, was mild and not clinically relevant. The combination of RIT with chemotherapeutic conditioning seems to be a therapy with an acceptable risk of treatment related morbidity and mortality as well as occurrence of severe acute GvHD.

Effect of Graft Engineering and Post-Transplantation Immunosuppression Regimen on Immunophenotypic Recovery in Recipients of Unrelated Donor Bone Marrow (T-Cell Depletion Trial) in a Multicenter Randomized Phase II-III Trial.

T cell depletion (TCD) of donor allogeneic blood or marrow grafts can reduce the incidence and severity of acute graft-versus-host disease (GvHD) but may place the patient at risk for disease relapse. This multicenter randomized trial examined the impact of selective depletion of αβ T cells (αβTCD) + cyclosporine immunosuppression in recipients of unrelated marrow grafts vs. T-replete marrow grafts + methotrexate and cyclosporine immunosuppression. Previously reported data from this trial have shown that 3-year disease-free survival (DFS) did not differ between these groups, although the αβTCD group showed improved neutrophil recovery, decreased incidence of acute GvHD, and reduced grade III-IV toxicities. In order to further clarify these findings, we examined the impact of each graft manipulation and immunosuppression strategy on lymphocyte subset recovery and associated outcomes. Lymphocyte recovery was assessed at 1, 3, 6, 12, 18, and 24 months post-SCT. Of all patients accrued, only patients that had survived for at least one year and who had at least four separate immunophenotyping assessments were included in this analysis (unmanipulated grafts = 66/206 32.0%; αβTCD grafts 57/203 28.1%). Non-parametric pair wise tests were used to compare recovery of absolute cell counts at each measurement period. We also fit a mixed model to each individual's data to estimate the rate of change in cell counts in the two groups. The slope estimate for each subject obtained from the mixed model was then used as a variable in models to assess DFS. The Cox proportional hazards test was used to examine associations between recovery of individual cell subsets and DFS. Recovery of total absolute CD3, αβ, γδ, CD3+CD4+, CD3+CD8+ T cell count as well as the absolute B cell count did not significantly differ between groups for at each measurement period. CD3+CD4+CD45RA naive T cell recovery remained below the expected adult normal range for all time points in both groups but was improved in patients who received unmanipulated grafts. Interestingly, NK cell absolute count recovery was significantly improved in patients that received TCD grafts over those who received unmanipulated grafts (p = 0.001) through the first year post-SCT. Analysis of the impact of individual cell subset recovery on DFS revealed that an absolute γδ T cell count greater than 1.75 x 105/ml at any time during the first year predicted for improved DFS regardless of treatment group (p = 0.05) consistent with previous single-institution reports. Although the αβTCD regimen did not result in improved DFS in this trial, these findings show that αβTCD is a reasonable graft engineering strategy that can reduce the incidence of acute GvHD without producing a significant negative impact on lymphocyte subset recovery. Indeed, patients in the αβTCD group showed markedly improved NK recovery. Approaches that are currently in early-phase trials such as post-SCT targeted T and/or NK cell therapies could be used to supplement the αβTCD regimen, potentially improving relapse-free survival.

Characterization of Ex Vivo Expanded Cord Blood Graft Treated with Chromatin Modifying Agents for Potential Clinical Use.

The lack of optimal number of repopulating hematopoietic stem cells (HSC) and graft failure following transplantation remain as obstacles to using cord blood (CB) as a true alternative graft for adult patients. Using chromatin modifying agents {5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA)}, we have developed a culture regimen that permits expansion of primitive CB CD34+ CD90+ cells resulting in a 10 fold expansion of in vivo repopulating cells without jeopardizing their multilineage differentiation capacity. Our results also indicate that the chromatin modifying agents likely mediate this function by inducing expression of endogenous genes (HoxB4, Bmi-1, GATA-2, etc.) previously implicated in self-renewal of HSC (Araki H et al. Blood 2007: 109:3670–3678). In our current studies we have further characterized the allostimulatory function of these expanded CB to assess its potential as grafts to mount an immune response. The percentage of 5azaD/TSA treated expanded CB cells co-expressing HLA-DR and CD86, or CD11c was less than cells expanded in cytokines alone. The expression of HLA-DR/CD40 was not altered following 5azaD/TSA treatment. In a mixed lymphocyte reaction (MLR), the irradiated CB cells (stimulator) were co-cultured with allogeneic peripheral blood T cells (responder) at 1:2 ratios. The MLR results indicate that 5azaD/TSA treated expanded CB grafts possessed diminished allostimulatory response as revealed by their relatively lower stimulation index in comparison to cells expanded in cytokines alone. Interestingly, the stimulation index of 5azaD/TSA treated cells (day 9) was even less than unmanipulated primary CB CD34+ cells (day 0). The reduced allostimulatory capacity of 5azaD/TSA expanded CB cells may favor in the reduction of incidence of graft rejection / graft failure following infusion. Alterations in cell fate regulatory mechanisms raise concerns for tumorigenesis, so the safety of such grafts was further assessed by examining the telomere length and cytogenetic analysis. Both cytogenetic analysis and examination of cell-cycle showed normal karyotype and diploid nature of expanded grafts, respectively. The telomere length of ex vivo expanded grafts as a putative indicator for their proliferative potential did not reveal significant alteration in their length in comparison to unmanipulated CB grafts. Also, in vivo repopulation assays with xenogeneic NOD/SCID mouse model showed no apparent tumor development following transplantation of ex vivo expanded 5azaD/TSA treated CB cells. In summary, our results indicate that the chromatin modifying agents not only result in expansion of in vivo repopulating CB cells but are capable of suppressing immunostimulatory capacity of expanded grafts and these grafts retain their telomere length and normal ploidy and can be expanded from a single unit for potential clinical use.

Retrospective evaluation of serum Epstein Barr virus DNA levels in 406 allogeneic stem cell transplant patients

An HLA-mismatched donor and a T-cell-depleted graft are known risk factors for Epstein-Barr virus (EBV) reactivation after stem cell transplantation. We studied the frequency and outcome of serum EBV DNA levels in patients transplanted with an unmanipulated graft from an HLA-identical donor. Overall, 5479 serial serum samples from 406 consecutive allogeneic stem cell transplant recipients were analyzed retrospectively for EBV DNA with quantitative polymerase chain reaction (PCR). EBV DNA was found in the serum of 56 of the 406 patients (14%). EBV positivity was seen in 9 % of the recipients of a graft from a sibling donor and in 29 % of those with an unrelated donor. EBV-PCR positivity resolved without specific treatment in a third of the cases, in another third the copy number increased progressively or was high in the last serum sample, and in the remaining third the copy numbers were low in all positive sera including the last sample. In multivariate analysis antithymocyte globulin given for any reason and grade III-IV acute graft-versus-host disease were the only statistically significant risk factors for EBV reactivation. Only 8/56 patients with EBV-DNA positivity were alive at the time of the present analysis. The outcome of EBV-PCR positivity could not be predicted by the copy number or the timing of the first positive sample. EBV reactivation was a common phenomenon in allogenic stem cell transplant recipients. In many patients the viremia resolved without EBV-directed treatment. Severe acute graft-versus-host disease and antithymocyte globulin given for any reason were risk factors for EBV viremia.

Adoptive Donor T-Cell Infusions Are Necessary and Efficient To Induce Lasting Molecular Remissions after Highly Purified CD34+ Sibling Donor Transplants for First Chronic Phase Chronic Myeloid Leukemia - Final Report and Long-Term Follow-Up of a Phase II Study.

Since September 1998, we prospectively studied the feasibility of transplantation using purified peripheral blood CD34+ cells from HLA-identical sibling donors in first chronic phase chronic myeloid leukemia (CML). A total of 60 patients (pts) with a median pretransplant risk score of 2 (range 1–4) has been included in this study. One patient received an unmanipulated graft due to poor CD34+ donor cell mobilization, while three pts (5%) were successfully retransplanted with an unmanipulated graft from the primary donor after secondary graft failure (n=2) or from an unrelated donor after hematologic relapse (n=1). As part of the study protocol, all pts were closely monitored for BCR-ABL transcripts using real-time RT-PCR analysis of peripheral blood cells as well as BCR-ABL-interphase FISH and metaphase karyotyping of marrow cells. Of the 60 pts, 56 were eligible for the application of donor lymphocyte infusions (DLI), but 7 pts did not receive DLI due to sustained molecular remission and complete chimerism. Thirty-one pts (52%) received DLI because of increasing BCR-ABL transcript levels or hematologic relapse, and 18 pts (30%) as programmed T-cell add-back. The median starting dose was 0.33 (0.01 – 10) x 106 CD3+ cells per kg with a median maximum dose 3.3 (0.17 – 100) x 106 CD3+ cells per kg. DLI induced a lasting reduction of median BCR-ABL transcript levels (BCR-ABL/GAPDH ratio) of more than 3 log10 and the estimate of being in a complete molecular remission at 7 years is 83% ± 5%. Six pts. (10%) did not respond to DLI, but 4 of these pts. attained a cytogenetic and molecular response by imatinib and/or interferon treatment. The cumulative risk of grades II-IV acute GvHD is 15% ± 5% for all study pts, and the risk of chronic GvHD is 25% ± 6%, respectively. After a median follow-up period of 46 (range 6 – 86) months for all pts, the cumulative 7-year survival estimate is 91% ± 4% (survival rate 92%). Causes of death were disease progression, secondary malignancy, liver failure, septicemia, and systemic capillary leak syndrome in one patient each. In conclusion, the concept of highly purified peripheral blood CD34+ cell transplantation in conjunction with adoptive DLI is associated with a particularly low risk of non-relapse mortality and allows induction of lasting molecular disease control in the majority of first chronic phase CML patients.

Long-term follow-up of gene-marked CD34+ cells after autologous stem cell transplantation for multiple myeloma

Gene marking can be used to investigate if progenitor cells harvested from patients are contaminated with tumorigenic cells. It can also provide information about the contribution of hematopoietic stem cells to long-term engraftment and about long-term transgene expression from integrated retroviral vectors. In order to study autologous-infused cell contribution to relapse as well as the long-term persistence of the transgene in hematopoietic cells following autologous bone marrow (BM) transplantation for multiple myeloma, we genetically marked autologous CD34+ enriched BM or peripheral blood cell grafts of eight myeloma patients using retroviral vectors. Six patients were subsequently transplanted with the marked graft and followed with regular time points of analysis. Briefly, mononuclear cells were harvested by leukapheresis during 2-4 consecutive days following priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF. The CD34+ cells separated on Cellpro ceprate avidin-biotin columns were exposed to the G1Na vector coding for neomycin resistance gene at a ratio of five vector particles per cell at three consecutive time points achieving an average transduction efficacy of 2% (0.43-5.1%). The patients were transplanted with a mixture of transduced cells and un-manipulated graft. Vector integration and transgene expression were analyzed by colony assays and polymerase chain reaction. The transgene could be detected for up to 5 years post-transplant in normal BM cells, even in remission following relapse and no side effects related to retroviral gene transfer were observed. There were no marked myeloma cells observed in the patients either in remission or in relapsing disease, which indicates that contribution of infused cells to relapse is unlikely.

Low mortality of children undergoing hematopoietic stem cell transplantation from 7 to 8/10 human leukocyte antigen allele-matched unrelated donors with the use of antithymocyte globulin

Human leukocyte antigen (HLA)-matched sibling donor hematopoietic stem cell transplantation (HSCT) is available for only approximately 30% patients needing HSCT. Use of alternative donors is associated with a high incidence and severity of graft-versus-host disease (GVHD). Here we report our experience with GVHD prophylaxis using pre-transplant rabbit antithymocyte globulin (rATG), in addition to post transplant cyclosporin A and methotrexate. Seventy-five children received unmanipulated grafts from 7 to 10/10 HLA allele-matched unrelated donors. Median follow-up was 25 months (range, 6-65 months). Only 2/75 patients (2.5%) developed acute GVHD grades III-IV, and 17/75 (25%) developed extensive chronic GVHD. Overall survival was 79%. It was similar in patients receiving grafts from 7 or 8/10 to 9 or 10/10 allele-matched donors, and similar in patients receiving peripheral blood stem cells and marrow. Six (11%) patients died owing to relapse, and 10 (13%) due to transplant-related complications. The addition of rATG appears to result in a low incidence of severe GVHD and overall mortality.

Can Only Partial T-Cell Depletion of the Graft before Hematopoietic Stem Cell Transplantation Mitigate Graft-versus-Host Disease While Preserving a Graft-versus-Leukemia Reaction? A Prospective Phase II Study

The study comprised 37 consecutive patients who underwent transplantation with a Campath-1H in vitro T cell–depleted granulocyte colony-stimulating factor–mobilized peripheral blood stem cell graft from an HLA-identical sibling, followed 24 hours later by an unmanipulated graft. Acute graft-versus-host disease (GVHD) was limited to grade I to II, whereas chronic graft-versus-host disease occurred in 9 patients, mostly (n = 7) with limited disease. Molecular relapses (8 chronic myeloid leukemia [CML] and 1 non-Hodgkin lymphoma) that occurred not earlier than the sixth month after transplantation were treated with donor lymphocyte infusion (DLI), which induced complete remission in all but 1 CML patient with persistent very low BCR-ABL molecular levels. With a median follow-up of 54 months (range, 29-84 months), the actuarial 5-year overall survival, disease-free survival, and transplant-related mortality are 78% (95% confidence interval [CI], 52%-88%), 78% (95% CI, 52%-86%), and 6% (95% CI, 1.5%-32%), respectively. All CML patients are alive and free of disease. The results of this prospective, nonrandomized study show that incomplete T-cell depletion in vitro with Campath-1H (in combination with DLI for molecular relapses in CML) may decrease the incidence of GVHD and transplant-related mortality with no adverse effect on disease-free survival. The described method decreases the number of T cells to an extent that severe GVHD is prevented while relapse is postponed to a time when the patient can be treated with DLI without severe side effects.

NK Cell KIR Reconstitution after Allogeneic Hematopoietic Cell Transplant (HCT) Is Affected by T Cell Number and Function.

NK cell KIR interactions are among the variables known to affect clinical outcomes including relapse, graft versus host disease (GVHD) and survival after HCT. We hypothesized that T cells in graft sources available for HCT may affect KIR recovery and the therapeutic potential of KIR alloreactivity. We studied KIR reconstitution (the percentage of KIR+ NK cells measured by flow cytometry) in blood collected from recipients at day +100 after T cell deplete (TCD-BMT) and unmanipulated (U-BMT) unrelated BM transplants. We found that KIR reconstitution was suppressed compared to the healthy donors, significantly more so after U-BMT transplants (donor: 48.42 ± 2.35% KIR+ NK cells versus recipient: 26.74 ± 1.94, n = 36; P < .001) than after TCD-BMT transplants (donor: 53.34 ± 3.25% versus recipient: 42.68 ± 3.32%, n = 38; P = .017), with P = .001 between the recipient groups. Additionally, multivariate Cox proportional hazards models showed that improved KIR recovery independently correlated with improved survival and that higher NK cell IFN-γ production independently correlated with more frequent acute GVHD in that patient cohort. These data suggested that T cell number in the graft affects KIR reconstitution and transplant outcome. We next examined other sources of hematopoietic cells in which T cell function may be suppressed either by growth factor mobilization (sibling donor unmanipulated peripheral blood: SibU-PB) or the innate naivety of the T cells (umbilical cord blood: UCB). KIR+ NK reconstitution on recovering cells at day +100 after all HCT graft types was significantly less than that on normal donor cells (normals 55.33 ± 1.73%, n = 124; all P < .0006). U-BMT recipients had significantly lower KIR+ NK recovery (27.31 ± 2.06%, n = 36 vs. SibU-PB: 37.58 ± 3.29%, n = 29; TCD-BMT: 42.68 ± 3.32%, n = 38; or UCB, 37.99 ± 2.54%, n = 49) when compared to all other transplant types. The highest absolute T cell inoculum, found in SibU-PB, showed KIR reconstitution similar to that of TCD-BMT, which had the lowest T cell content (p=0.29), perhaps due to the lower alloreactivity of the Sib grafts and to the G-CSF-priming which preferentially mobilizes T cells with a suppressive phenotype. Similarly, KIR reconstitution was better after UCB compared to U-BMT (P = .0027), possibly due to the more permissive interactions with naive T cells. These results suggest that reduced T cell number after T cell depletion, suppressed T cells found after growth factor mobilization, or naive T cells present in UCB grafts enhance in vivo KIR reconstitution after allogeneic HCT when compared to unmanipulated marrow grafts. Such enhanced KIR reconstitution may have clinical consequences. Graft T cells may directly compete for cytokines and growth factors, or may be a surrogate marker for other transplant factors such as the development of GVHD and the requirement for intensive post-transplant immunosuppression. Understanding these interactions will allow judicious selection of hematopoietic cell source to select for enhanced KIR recovery. For example, among unrelated unmanipulated donor grafts, KIR+ NK recovery was significantly better using UCB than adult donors and further investigation may show that this is advantageous to improve clinical outcomes.

Polymorphisms of NOD2/CARD15 Are Associated with Clinical Outcome in T-Cell Depleted HLA-Identical Sibling Allogeneic Stem Cell Transplantation.

Three single nucleotide polymorphisms (SNP) in NOD2/CARD15 have been associated with the incidence and severity of acute GVHD following allogeneic stem cell transplantation (allo-SCT). The potential clinical influence of SNP in NOD2/CARD15 in a model of allo-SCT with a low incidence of GVHD, such as T-cell depleted transplants (TCD), has not been studied. We analyzed the association of SNP in NOD2/CARD15 with the outcome of 84 patients undergoing allo-SCT with TCD by means of CD34+ selection. Results were compared with a concurrent group of 94 patients receiving an allo-SCT with unmanipulated grafts (Non-TCD). Transplants were performed in a single institution, donors were HLA identical siblings and all patients received myeloablative-conditioning regimen. Median follow-up was of 30.6 months (range, 0.2–112). SNP 8, 12 and 13 were analyzed by RT-PCR allelic discrimination method in all patients and in 137 donors. SNP in NOD2/CARD15 were detected in 13.8% of patients and in 10.7% of donors. Results of the association of patient's NOD2/CARD15 genotype and outcome are given in the table. In TCD transplants, independent risk factors associated with event free survival in a multivariate analysis were: patient's NOD2/CARD15 genotype (RR 2.7, p=0.02), patient's age (RR 2.3, p=0.03) and conditioning with radiotherapy (RR 2.7, p=0.03). The only association of donor's NOD2/CARD15 genotype with clinical outcome in the multivariate analysis was with acute GVHD (RR 3.3, p=0.03). In conclusion, polymorphisms in NOD2/CARD15 have a strong association with clinical outcome in TCD transplants, which is independent of GVHD.Overall group (n=178) SNP vs. WTP*TCD (n=84) SNP vs. WTP*Non-TCD (n=94) SNP vs. WTP*Acute GVHD II–IV27 vs. 22N.S22 vs. 40N.S33 vs. 12N.SChronic GVHD61 vs. 350.129 vs. 14N.S100 vs. 440.005Event free survival21 vs. 470.0317 vs. 480.0422 vs. 31N.SOverall survival30 vs. 590.0133 vs. 560.0430 vs. 48N.SWT, wild type; N.S, non significant; * Log-rank test

Increased Risk for Treatment-Related Mortality of Bone Marrow Transplantation in GSTM1-Positive Recipients.

Treatment-related mortality (TRM) is a major obstacle to successful allogeneic hematopoietic stem cell transplantation (HSCT). A variety of drugs are used in allogeneic HSCT, such as chemotherapeutic agents, immunosuppressants, antibiotics, antifungal and antiviral drugs, thus, a genetic polymorphism in metabolic enzymes would affect the metabolism of drugs and subsequent TRM after HSCT. We recently reported a metabolic enzyme UDP glycosyltransferase 2 family, polypeptide B17 (UGT2B17) has a null phenotype, and the use of a UGT2B17-positive donor is an independent risk factor for higher TRM and lower survival after HSCT (ASH annual meeting 2004, abstract# 1837). Here, we focused attention on glutathione S-transferase (GST) M1 and GSTT1 enzymes. These enzymes mainly metabolize chemotherapeutic agents, chemical carcinogens and by-products of oxidative stress and are absent from more than 50% of some populations. To assess the significance of homozygous GSTM1 and GSTT1 gene deletion in allogeneic HSCT, we analyzed DNA from 373 patients with a hematological disease and their HLA-A, B, C, DRB1-identical unrelated bone marrow donors using PCR method. All patients received an intensive myeloablative pretransplant conditioning regimen, an unmanipulated marrow graft, and cyclosporine A or tacrolimus as GVHD prophylaxis. Homozygous GSTM1 and GSTT1 gene deletion were observed in 56% and 45% of patients with a hematological disease, respectively, and 57% and 46% of healthy donors, respectively. There was no difference in the frequencies between disease group and donor group. We next applied Cox proportional hazard model to multivariate analysis for TRM which was defined as any death that occurred while the patient was in remission, acute GVHD, chronic GVHD, relapse, disease-free survival (DFS) and overall survival (OS). The analysis showed no significant association between GSTT1 polymorphism and any outcome. However, GSTM1-positive recipient was significantly associated with higher TRM and lower survival: TRM; Odds ratio (OR), 1.49; 95% confidence interval (CI), 1.03–2.16; P=0.036: DFS; OR, 1.50; 95% CI, 1.08–1.97; P=0.013: OS; OR, 1.41; 95% CI, 1.04–1.92; P=0.030. TRM was analyzed in relation to GSTM1 genotype in the patient by the Kaplan-Meier method (Fig 1). TRM in the GSTM1-positive patients (42.9%, n=166) was significantly higher than that in the GSTM1-negative patients (29.3%, n=201) (P=0.04). These data suggest the presence of the GSTM1 null genotype in recipient provide better protection against TRM after allogeneic HSCT. Some reactive intermediates or toxic metabolites generated by GSTM1 may initiate or promote the development of TRM. [Display omitted]

Cytomegalovirus Reactivation in Pediatric Hemopoietic Progenitors Transplant

Cytomegalovirus (CMV) disease is an important infectious complication after allogeneic hemopoietic progenitors transplant (HPT), but few data are available in children. To investigate the factors influencing CMV reactivation, subsequent relapses of positive antigenemia, and the development of organ disease in children, the authors retrospectively analyzed 108 children who received allogeneic HPT for malignant conditions at one center. Of these, 41 were CMV serology positive (donor or recipient) before HPT. All those with CMV-positive serology received high-dose acyclovir in conjunction with weekly CMV phosphoprotein-pp65 antigen monitoring of the peripheral blood during the first 3 months. Those with CMV reactivation (positive antigenemia) received preemptive treatment with ganciclovir and/or foscarnet to prevent CMV disease. The incidence of positive antigenemia in this cohort was 41.5% at a mean of 44 +/- 31.6 days after HPT. Two patients (4.9%) subsequently developed late CMV disease. Recipient CMV status was significantly (P = 0.0001) more relevant to reactivation than donor status. Reactivations were significantly more common in single recipient seropositive than double (donor and recipient) positive pairs (P = 0.05). Reactivations were significantly more common in recipients of unrelated donor grafts than matched-related donor grafts (P = 0.025). Reactivations also occurred significantly more in T-cell-depleted graft recipients (P = 0.004) than recipients of unmanipulated grafts. The subsequent development of disease was more common in a CMV-seropositive recipient receiving a CMV-seronegative, T-cell-depleted, unrelated donor graft, and after transplantation receiving treatment for acute graft-versus-host disease. Patients with identified high risk factors for CMV reactivation and disease should be monitored by CMV PCR, and early preemptive treatment should be instigated to prevent the development of disease.

Occurrence of autoantibodies in chronic graft vs. host disease after allogeneic stem cell transplantation

Chronic graft vs. host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Chronic GVHD (cGVHD) has many similarities to de novo autoimmune disorders. While the presence and association of autoantibodies is well reported in these disorders, their role and clinical use remains a less studied area after SCT. We report the presence of autoantibodies in SCT recipients and a possible association with presence of cGVHD. During routine follow-up visits peripheral blood samples were tested for: rheumatoid factor (RF), antinuclear antibody (ANA), double stranded DNA (dsDNA), antimitochondrial antibody, antismooth muscle antibody (Anti Sm), antiendomysial, antireticulin antibodies, antithyroid peroxidase antibodies and an extractable nuclear antigen screen, in 13 SCT recipients. Six of 13 (46%) patients had one or more autoantibodies. All the patients with antibodies had cGVHD where as none of the patients without cGVHD had any autoantibodies (P = 0.025). Three (23%) patients had only one autoantibody and three (23%) of them had more than one autoantibody. ANA was positive in three (23.3%) patients, double stranded DNA in four (30.7%) patients, RF in one (7.6%) and Anti Sm muscle in two (15.3%) patients. In the present study, autoantibodies were detected predominantly in patients with presence of cGVHD. They also appeared to be more frequent in an unmanipulated graft and so less in patients with a T-cell depleted allograft. In two of 13 patients only there appeared to be an association between the antibody titre and flare up in skin symptoms. In conclusion, this small series raises interesting questions about the presence and role of autoantibodies after SCT and their association with cGVHD.

Economic analysis of unrelated allogeneic bone marrow transplantation: results from the randomized clinical trial of T-cell depletion vs unmanipulated grafts for the prevention of graft-versus-host disease

Unrelated-donor marrow transplantation is a potential option for transplant candidates lacking a compatible related donor. The T-cell Depletion Study compared the 3-year disease-free survival for patients receiving T-cell-depleted (TCD) donor marrow (n = 203) vs unmanipulated donor marrow with methotrexate and cyclosporine (M/C) (n = 207). Hospital costs during index admission were documented with billing data, while hospital costs during subsequent 6-month follow-up were estimated from case report forms. Patients with index admission billing were included in the analysis (TCD = 119, M/C = 127). Total hospital length of stay (LOS) was similar across groups, with medians 47.0 days for TCD and 52.0 days for M/C (P = 0.72). Total hospital costs were comparable, 145,115 dollars vs 141,981 dollars (P = 0.63) for TCD and M/C, respectively. However, controlling for site and patient characteristics, TCD was associated with a 12.1% reduction in LOS for the index admission (95% CI -19.4%, -4.3%). Independent of treatment, HLA matching (6/6) was associated with an 8.6% (95% CI -17.4%, +1.2%) reduction in the index admission LOS, while cost was lower by 15.8% (95% CI -26.7%, -3.3%). Treatment costs were similar for TCD and M/C study groups. Savings on reduced cost for treating acute graft-versus-host disease were likely offset by increase in serious infections in the TCD arm.

Autologous peripheral blood stem cell transplantation with in vivo T-cell depletion for life threatening refractory systemic lupus erythematosus

We report the first Australian application of autologous haemopoietic stem cell transplantation in a 39-year old woman with severe systemic lupus erythematosus (SLE) and multiple life threatening complications, refractory to conventional therapy including intravenous cyclophosphamide. Our transplant technique, although not unique, differs from most published reports, in which an unmanipulated peripheral stem cell graft was used with in vivo lymphocyte depletion using rabbit antithymocyte globulin (ATG). Successful stem cell mobilization was achieved using granulocytecolony stimulating factor mobilization with methylprednisolone cover, after an initial attempt at mobilization was curtailed by respiratory arrest from upper airway obstruction due to cricoarytenoiditis, requiring tracheostomy. Conditioning regimen for the transplantation was cyclophosphamide 50 mg/kg on days -5 to -2 and rabbit ATG 2.2 mg/kg on days -3 and -2. An unmanipulated autograft was infused, with in vivo T-cell depletion achieved through a further dose of ATG given on day +2 postinfusion. The autologous transplant was well tolerated without fever or other serious complication. At 12 months follow-up post-transplantation, there is an objective evidence of near-complete response with SLE disease activity index scores falling from 40 pretransplant to 2. We conclude that HSCT with unmanipulated peripheral stem cell graft and in vivo lymphocyte depletion with ATG is safe and effective therapy for cyclophosphamide refractory SLE.

Donor's Mannan-Binding Lectin (MBL) Gene Polymorphism Is Associated with Invasive Fungal Infection Following Allogeneic Stem Cell Transplantation.

Despite improvement in diagnosis and therapy, invasive fungal infections (IFI) remains one of the most important barriers to the success of allogeneic stem cell transplantation (allo-SCT). Mannan-binding lectin (MBL) gene polymorphisms have been associated with an increased risk of infections, particularly in immunocompromised patients. We analyzed the association of MBL gene polymorphisms with the incidence of IFI in 138 HLA identical donor-patient pairs, undergoing allo-SCT in our institution between March 1995 and December 2003. Median age at time of transplantation was 38 years (range 17 – 63); 73 (53%) patients were male. Diagnoses were AML (n=43), CML (n=23), ALL (n=19), NHL (n=18), and other hematological malignancies (n=35). 72 (52%) patients were in early phase of the disease and 66 (48%) in advanced phase. 129 (93%) donors were siblings and 9 (7%) unrelated. The source of stem cells was peripheral blood in 133 (96%) patients and bone marrow in 5 (4%). Conditioning regimen was myeloablative in 126 (91%) and non-myeloablative in 12 (9%). 71 patients (51%) received a T cell depleted graft by CD34+ selection, and in 67 (49%) cases the graft was unmanipulated. Six single nucleotide polymorphisms (−550 G/C, −221 C/G, 4 C/T, 52 CGT/TGT, 54 GGC/GAC and 57 GGA/GAA) in the MBL2 gene were genotyped using PCR and sequence specific primers. 22 (16%) donors and 21 (16%) patients of the 138 pairs carried O/O, LXA/LXA or LXA/O genotypes, which are related to a low production of MBL protein. After a median follow up of 21 moths, 25 (18%) patients developed an IFI, with an overall actuarial probability for this complication of 22%. In the univariate analysis, factors associated with a higher probability of IFI were: unmanipulated grafts (10% in CD34+ selected vs. 27% in unmanipulated grafts, p=0.001), aGVHD II–IV (9% vs. 30%, p=0.0007), and “low-producing” MBL donor genotype (14% vs. 41%, p=0.01). The group of patients with aGVHD II–IV and with a “low-producing” MBL donor genotype had an actuarial probability of IFI of 78% vs. 18% for the remaining patients (p=0.0003). No association between recipient's MBL polymorphism and IFI was found. All variables with a p value < 0.2 in the univariate analysis were introduced in the multivariate analysis. Only two independent factors were associated with a higher incidence of IFI in the multivariate analysis: “low-producing” MBL donor genotype (RR 7.2, p=0.007) and aGVHD II–IV (RR 5.5, p=0.02). In conclusion, donor's MBL gene polymorphism possesses strong predictive value for IFI. This finding could be useful for selecting the most appropriate donor. In addition, studies investigating primary anti-fungal prophylaxis in patients developing aGVHD II–IV and with a “low-producing” MBL donor genotype are warranted.

Allogeneic Stem Cell Transplantation for Children with Advanced Primary MDS: Results from the EWOG-MDS Study Group Employing a Pre-Transplant Preparative Regimen with Busulfan, Cyclophosphamide and Melphalan.

MDS in children is a rare disorder characterized by dysplasia and defined genetic abnormalities. In most patients (pts) MDS arises without known predisposing conditions (primary MDS). Here, we report the results of 55 males and 30 females with advanced primary MDS enrolled in the prospective EWOG-MDS trial 97. Data were analysed according to the most advanced FAB-type prior to SCT: 32 pts were classified as RAEB, 40 as RAEB-t and 13 as myelodysplasia-related AML (MDR-AML). Median age at diagnosis was 9.5 yrs (0.1–17.6) and median time from diagnosis of advanced MDS to SCT 4 mo (0.5–31). Cytogenetics revealed monosomy 7 in 32 pts, trisomy 8 in 7, a complex karyotype in 9 and other abnormalities in 9; karyotype was normal in 26 pts and unknown in 2. 31 pts had received AML-like therapy prior to SCT. All pts were given an unmanipulated graft after condititioning with busulfan 16 mg/kg, cyclophosphamide 120 mg/kg and melphalan 140 mg/m. Source of stem cells was bone marrow in 56 pts, peripheral blood in 25, cord blood in 2 and unknown in 2. 36 pts were transplanted from an HLA-identical relative (MFD), 49 pts from an HLA-identical or 1-antigen disparate unrelated donor (UD). GVHD prophylaxis consisted of CSA alone for MFD, whereas recipients of a UD graft generally received CSA, methotrexate and anti-lymphocyte globulin. Two pts suffered graft failure. The cumulative incidence of grade II-IV acute GVHD and chronic GVHD was 40% (SE 5%) and 25% (SE 5%), respectively. 18 pts suffered transplant-related mortality (TRM), the cumulative incidence of TRM in pts grafted from a MFD or UD being 14 and 25%, respectively (p=n.s.). Presence of acute GVHD II-IV (p<0.01), spleen size at SCT ≥ 1 cm below the costal margin (p=0.03) and age ≥ 12 years (p=0.04) predicted an increased risk of TRM. 17 patients relapsed at a median of 12 mo after SCT (1–107). The 5-year probability of leukemia recurrence was 29%, with no difference between MFD and UD transplants. While the highest FAB type prior to SCT predicted relapse with a cumulative incidence rate increasing from RAEB (13%, SE 10%) to RAEB-t (24%, SE 11%) and MDR-AML (69%, SE 20%) (p=0.03), the use of intensive chemotherapy prior to SCT or blast percentage at SCT did not. With a median observation time after SCT of 29 months (6–107) the EFS at 5-years was 62% (SE 12%) and 39% (SE 11%) for pts given SCT from a MFD or an UD, respectively (p=n.s). These results indicate that a large proportion of pts with advanced MDS can be rescued by SCT. Disease recurrence remains the main cause of treatment failure. Intensive chemotherapy prior to SCT should not be routinely employed.