Single Antigen Mismatch Research Articles

Excellent Outcomes of HLA Matched Related Donor Transplant for Adults with Severe Sickle Cell Disease Using a Non-Myeloablative Conditioning with Thiotepa and Post-Transplant Cyclophosphamide: Multi-Center International Experience

Introduction: The validated curative treatment for sickle cell disease (SCD) is allogeneic hematopoietic stem-cell transplantation (HSCT). Despite paucity of available matched sibling donors (10-14%), they are preferred due to less immunogenic complications like graft versus host disease (GVHD) and graft failure (GF) using alternative donors. In-vivo T-cell depletion by post-transplant cyclophosphamide (PTCy) has been shown to be most effective in GVHD control and a non-myeloablative regimen minimizes the toxicity associated with HSCT in this vulnerable population. In 2013, the multi-institutional Vanderbilt Haploidentical Learning Collaborative showed the improved outcomes of their non-myeloablative regimen for related haploidentical BMT for SCD (ClinicalTrials.gov identifier NCT01850108). We used a similar non-myeloablative approach to improve donor engraftment rates, while minimizing GVHD. Material and methods: This study was conducted at three international centers using similar conditioning from our experience in multi-institutional Vanderbilt Global Haploidentical Learning Collaborative to optimize haploBMT for SCD. The conditioning regimen included ( Figure3): rabbit anti-thymoglobulin (ATG) 4.5 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, cyclophosphamide 29 mg/kg and total body irradiation (TBI) 200 cGy. GVHD prophylaxis included PTCy 100 mg/kg, mycophenolate mofetil (MMF), and sirolimus. Stem cell source included bone marrow (BM) mobilized with filgrastim (5-10 μg/kg/d x 3-5 days) and peripheral blood (PB). Primary graft failure (GF) was defined as <5% donor myeloid/lymphoid chimerism or <5% donor cells by whole blood (WB) chimerism by day+28 posttransplant. Secondary graft rejection was defined <5% donor myeloid/lymphoid chimerism or <5% donor cells by whole blood (WB) chimerism with prior documentation of >5% donor cells by day +28. The Event free survival (EFS) was defined by survival with no GF, no death and/or no chronic GVHD. This cohort includes 4/22 patients (18.2%) had prior graft failure from a previous bone marrow transplants. Results: Here we report the outcome of 22 SCD patients (100% HbSS genotype), with a median age of 26.5 (15-41), transplanted from 10/10 MRD 86.4 % (n= 19) or single antigen mismatch related donor (MMRD) (9/10) 13.6% (n=3). The stem cell source was PB in 54.5% (12/22), and BM in 45.5% (10/22). Median CD34 stem cells dose was 6 X 10 6/Kg (1.2 - 10.25). Neutrophils and platelets engrafted at a median of 21.0 (15 - 26) and 25.0 (12 - 45) days, respectively. Demographic and clinical characteristics of participants summarized in Table 1. No primary or secondary graft failure was observed in the participants based on the last follow-up and time of analysis of this report. For participants with durable engraftment, the median WB chimerism values for D+28 and D+365 engraftment was 99.25% (n=14; IQR: 52 - 100) and 96.5% (n=16; IQR: 52 - 100), respectively. The median follow-up time was 14.6 months (n=22; IQR: 0.5-45.44). The overall incidence of acute GVHD grade III-IV was 4.5% (n= 1/22), and no incidence of chronic GVHD. There was no transplant related mortality. A Kaplan-Meier based probability of overall survival (OS) was 100%, Figure 1. All patients were off immunosuppression at 1-year post transplant. EFS as defined in the method was 100%, Figure 2. The EFS of free acute GVHD grade III-IV was 95.5%. Conclusion: This result shows the superiority of Thiotepa based non-myeloablative conditioning and PTCy for MRD transplants in adults with severe SCD. It not only minimizes the toxicity and GVHD but provides optimal donor engraftment mitigating against risk of secondary myeloid neoplasms from mixed chimerism. This needs further validation in a larger cohort.

Comparison of HLA Allele Mismatch and Antigen Mismatch in Unrelated Bone Marrow Transplantation in Patients with Leukemia

We compared the effect of HLA single-antigen and single-allele mismatched unrelated bone marrow transplantation (UBMT) without in vivo/ex vivo T cell depletion. Becasue a single DRB1 mismatch is preferred among 1-allele or 1-antigen mismatched donors, we performed mismatched allele- or antigen-specific analyses with a single DRB1 mismatch as the reference. In adjusted comparison by multivariate analyses, an HLA-DRB1 single-allele mismatch resulted in a decreased risk of nonrelapse mortality (NRM; relative risk [RR], 1.33; 95% confidence interval [CI], 1.08 to 1.63, P = .006) compared with an HLA-DR single-antigen mismatch and conferred a decreased risk of NRM (RR, 1.25; 95% CI, 1.01 to 1.57; P = .025) and overall mortality (RR, 1.16; 95% CI, 1.00 to 1.37; P = .046) compared with an HLA-C single-antigen mismatch. Relative to an HLA-DRB1 single-allele mismatch, 2-mismatch transplants, including those with 1 or more antigen mismatches, resulted in a significantly increased risk of NRM (1-antigen/1-allele mismatch: RR, 1.68; 95% CI, 1.03 to 2.05; P < .001; 2-antigen mismatch: RR, 1.58; 95% CI, 1.04 to 2.02; P = .001) and overall mortality (1-antigen/1-allele mismatch: RR, 1.27; 95% CI, 1.09 to 1.47; P = .002; 2-antigen mismatch: RR, 1.27; 95% CI, 1.03 to 1.57; P = .02). NRM correlated with the combined number of mismatches and allele or antigen mismatches, with rates of 22%, 27%, 32%, 31%, and 38% at 4years for full match, single-allele mismatch, single-antigen mismatch, 2-allele mismatch, and 2 mismatches that included an antigen mismatch, respectively. Our results support the preference for an allele mismatch rather than an antigen mismatch in unrelated bone marrow donors with 1 DR mismatch or 2 mismatches for T cell–replete UBMT.

Extended CCR5 Blockade in Graft-Versus-Host Disease Prophylaxis – a Phase II Study

Background: Blocking lymphocyte trafficking after allogeneic stem cell transplantation (alloSCT) may prevent graft-versus-host disease (GvHD) without interfering with graft-versus-tumor (GvT) activity. We previously reported that brief (up to day+30) CCR5 blockade using maraviroc (MVC, Pfizer) after reduced-intensity conditioned (RIC) alloSCT resulted in a low incidence of acute GvHD and absence of early liver and gut GvHD, although delayed GvHD still occurred. We designed a phase II study to test the hypothesis that extended administration of MVC would be feasible, safe and provide protection against late-onset GvHD without impairing immune reconstitution or GvT responses.Patients and Methods: In April 2013 we initiated a 37-patient (pt) phase II study to test an extended course of MVC in recipients of RIC alloSCT from unrelated donors. Pts receive fludarabine 120 mg/m2 and busulfan i.v. 6.4 mg/kg followed by peripheral blood stem cells. MVC 300 mg b.i.d. is orally administered from day -3 to day +90 in addition to standard prophylaxis with tacrolimus and methotrexate. The primary endpoint is the cumulative incidence of grade 2-4 acute GvHD by day 180. As of July 2014 we enrolled 20 pts at high risk for transplant-related toxicity by virtue of age (median=64, range 55–72), donor source (matched unrelated 80%, single-antigen mismatch unrelated 20%) or comorbidities (comorbidity index: low 15%, intermediate 35%, high 50%). Underlying diseases were AML (16), MDS, MPD, ALL and CTCL (1 each).Feasibility and Safety: The median follow-up on surviving patients is 5.7 months. The 3-month course of MVC was well tolerated with no increased toxicity; two pts did not complete their treatment due to early disease relapse and one patient discontinued therapy due to a skin reaction with eosinophilia where the histological features favored a drug reaction and the attribution to MVC was possible. Postural hypotension, a known dose-dependent toxicity, was observed in one pt who completed the course with a 50% dose reduction.Engraftment and Immune Reconstitution: The median time to ANC>500/μL was 12 d (range 10-21) and platelets>20k/μL was 14 d (range 9-28). The median whole blood and T-cell donor chimerism levels at day 100 were 95% (range 12–100%) and 80% (range 23–94%) respectively, which are similar to historical rates. Median CD4 counts on day 30 were 341 (range 206-424). Only 3/16 evaluable pts had Ig levels<500 mg/dL in the first 100 days.GvHD: Sixteen pts are evaluable with > 3 mo of follow-up. The day-180 cumulative incidence rates (± s.e.) of grade 2-4 and grade 3-4 acute GvHD are 25 ± 11% and 6 ± 6% respectively (Fig. 1). Of patients who developed acute GvHD in the first 180 days, there have been no cases of liver GvHD, 2 cases of stage 1 steroid-responsive gut GvHD and 1 case of severe diarrhea with combined features of GvHD and leukemic infiltrates in the gut. These results are comparable to the GvHD rates in our phase I/II MVC study (grade 2-4: 23.6% and grade 3-4: 5.9%), which included related and unrelated donor transplants. These results also compare favorably with a 45% day-180 acute GvHD rate seen in similar patients treated with our standard GvHD prophylaxis alone. Notably, there has been no treatment-related mortality. Five patients have relapsed at a median of 2.6 months post-transplant (range 0.93 – 3.5), which is similar to our historical rates after RIC alloSCT.PD analysis: We developed a phosphoflow assay to assess in real-time the activity of MVC in fresh blood samples. The assay quantifies the activation of CCR5 by measuring the phosphorylation of C-terminal serine residues as a result of CCL4 stimulation. In 15 evaluable patients, we observed diminished pCCR5 levels with CCL4 stimulation on day 0 as compared to day -6 (Fig. 2).In summary, our preliminary results support the feasibility, safety and protective activity of the CCR5 antagonist MVC against acute GvHD, with preferential activity against visceral GvHD. Continued pt enrollment and follow-up are ongoing. Updated safety, efficacy and PD results will be presented. A multi-center study (BMT-CTN 1203) will be initiated later this year to further clarify the role of this novel strategy in improving the outcome of alloHSCT. [Display omitted] [Display omitted] DisclosuresReshef:Pfizer: Research Funding. Off Label Use: Maraviroc for graft-versus-host disease prophylaxis.

Comparable Outcome After Single-Antigen Mismatched Versus Matched Unrelated Donor Hematopoietic Cell Transplantation,

Abstract 4165 Introduction:Hematopoietic cell transplantation (HCT) is the treatment of choice for many hematological malignancies and often the only curative option. Despite continuous expansion of unrelated donor registries worldwide, not all patients have a matched donor available and single antigen mismatched donors are used for patients at high risk of relapse or progression. However, it remains unclear whether or not single-antigen mismatched and matched unrelated donor transplantations generate comparable clinical outcomes. Here, we present the results of a unicentre analysis approaching this question. Patients and Methods:The outcome of all patients transplanted from unrelated donors between 2000 and 2009 at the University Hospital in Leipzig was analyzed. A total of 206 patients with a median age of 38 (range 18–58) years with acute leukemias, chronic myeloid leukemia, myelodysplastic syndrome or non Hodgkin‘s lymphoma were treated with a myeloablative regimen consisting of 12 Gy fractionated total body irradiation (n=189) or busulfan 16mg/m2 (n=17) in combination with cyclophosphamide. All patients received antithymocyte globulin during the conditioning regimen and graft-versus-host prophylaxis with cyclosporine and short course methotrexate. Donors were considered matched according to the typing available at the time of transplantation: Antigen typing in class I and allele typing in class II was available prior to 2006 and 10/10 4 digit HLA typing thereafter. Donors were considered matched if no HLA-antigen mismatch was detected. One hundred and fifty five patients were matched for the HLA loci A, B, C, DRB1 and DQB1 at the antigen level, while 39 patient/donor pairs had a single antigen mismatch and 12 a mismatch of two or more antigens. Disease stage, comorbidity index and Gratwohl score were well balanced in both groups. Results:After a median follow-up of 49 months, 54% of the 206 patients were alive. Most interestingly, there was no difference in overall survival (OS) at 5 years between HCT with matched and HCT with mismatched donors [52% vs 49% respectively (p=0.48)]. Also similar were event free survival (EFS) at 47% vs. 39% (p=0.44), relapse incidence (RI) 34% vs. 50% (p=0.22) and non-relapse mortality (NRM) 28% vs. 22% (p=0.81) in the matched versus mismatched donors. Acute graft versus host disease (GvHD) grade one or two occurred in 58% of all patients, while 14% had grade three or four and 28% had no signs of acute GvHD. The incidence of GvHD was comparable after antigen mismatched and antigen matched HCT. As expected, there was a significantly better OS, PFS and lower NRM for patients with early (n=104) vs. advanced disease (p=0.02) and patients with high resolution typing vs. low resolution typing (n=99; P=0.04). Even within these subgroups, however, the use of a single antigen mismatched donor did not worsen the results. Conclusion:In this unicenter analysis a comparable outcome was found after single antigen mismatched HCT compared to matched unrelated donor HCT. An antigen mismatched donor seems to be an acceptable option for patients with high risk malignant disease for whom no fully matched donor is available Disclosures:No relevant conflicts of interest to declare.

Whole Blood Donor Chimerism At Day 30 After Reduced Intensity Conditioned Allogeneic Stem-Cell Transplantation Predicts Disease Relapse, and Is Strongly Associated with Pretransplant Lymphodepletion

Abstract 1939 Background:RIC SCT relies heavily on graft-versus-tumor alloreactivity, and relapse remains a major barrier to a favorable outcome. Early prediction of relapse would allow early intervention such as donor lymphocyte infusions (DLI), but factors that predict relapse and methods for detection of minimal residual disease are often disease specific and not standardized. The level of donor-recipient chimerism has been associated with both graft rejection and relapse, but the optimal timing, desired level, and predictive value of chimerism testing in RIC SCT are unclear. Furthermore, the clinical utility of whole blood (WB) vs T-cell chimerism is not well defined, particularly after RIC SCT. Methods:We aimed to assess the predictive value of early WB and T-cell chimerism on the incidence of relapse in SCT pts receiving a uniform RIC regimen for hematologic malignancies. Between August 2006 and May 2011, 120 consecutive patients (pts) underwent allogeneic SCT following conditioning with fludarabine 120mg/m2 and busulfan i.v. 6.4 mg/kg. Pts were not treated with pre-emptive DLI, but could receive DLI for relapse. Hematopoietic chimerism was determined by DNA genotyping of short tandem repeats. Chimerism was determined on WB and then on enriched T-cells, obtained by selection using CD3-labeled magnetic beads. We conducted a cumulative incidence analysis of relapse, using day 30 as a landmark to determine the predictive properties of chimerism studies obtained at that time. Results:The 1-yr cumulative incidence of relapse in this cohort was 48.3 ± 4.7%, and the median time to relapse was 102 days (range 16–566 days), highlighting the importance of early monitoring. 68 pts were in remission and had evaluable chimerism data on day 30; importantly, the 1-year incidence of relapse was not different between pts who did and did not have day 30 chimerism measured (50.2 ± 6.5% vs. 42.9 ± 7.2%, P=0.45). Median follow up was 238 days (range 75–1420). Median age was 61 (range 21–76) and 56% were male. Underlying diseases were AML (24), MDS (13), NHL (10), myelofibrosis (6), CTCL (4), Hodgkin (3), myeloma (3), CLL (2), aplastic anemia (2), CML (1). Pts received a peripheral blood stem cell graft (67) or bone marrow (1), harvested from a matched related (30) or unrelated (38) donor. A single antigen mismatch was present in 6 cases. GVHD prophylaxis was tacrolimus (55) or cyclosporine (13) based.Median WB donor chimerism at day 30 was 96% (range 31–100%). A cumulative incidence analysis of relapse from day 30 revealed that day 30 WB chimerism had a significant association with relapse (HR 0.97, 95% CI [0.95–0.99], P=0.0011), reflecting a 3% decrease in risk of relapse for each 1% increase in chimerism. Risk of relapse according to different day 30 chimerism levels is displayed in figure 1. Using a day 30 chimerism cutoff of 95%, we found a significant association with incidence of relapse (HR 0.29, 95% CI [0.15–0.57), P=0.0003). Pts who were alive without relapse at the end of follow-up had a significantly higher chance of >= 95% day 30 WB chimerism compared to pts who relapsed (88.9% vs. 45.5%, P=0.002). In 38 pts with myeloid diseases, there was a significant association between day 30 WB chimerism and risk of relapse (HR 0.97, 95% CI [0.96–0.99], P=0.00065), while in 22 pts with lymphoid diseases, an association did not reach statistical significance (HR 0.93, 95% CI [0.84–1.00], P=0.09).Median T-cell chimerism was 70% in the 49 evaluable pts with these measurements (range 26–99%). T-cell chimerism on day 30 did not predict relapse (HR 1.0, 95% CI [0.98–1.02], P=0.95).We analyzed potential associations between day 30 WB chimerism and various transplant and patient characteristics. A higher chimerism level was significantly associated with lower day 0 lymphocyte count (P=0.004) and lower preconditioning lymphocyte count (P=0.01), but was not associated with recipient or donor age, cell doses, busulfan levels, disease type and donor type. The day 0 lymphocyte count was also a strong predictor of relapse (HR 6.87, 95% CI [6.56–7.04], P=0.00035). Conclusions:Whole blood and not T-cell chimerism at day 30 is predictive of relapse after RIC SCT. WB chimerism is strongly associated with lower lymphocyte counts before and after the conditioning regimen. These data highlight the importance of adequate lymphodepletion and can be useful in designing future trials testing pre-emptive interventions to prevent relapse after RIC SCT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Inhibition of Lymphocyte Trafficking Using a CCR5 Antagonist – Final Results of a Phase I/II Study

Abstract 1011Inhibition of lymphocyte trafficking early after allogeneic stem cell transplantation (SCT) may prevent GvHD without interfering with GvL activity. Animal models and genomic data in humans indicate that the interaction between CCR5 and its ligands CCL3, CCL4 and RANTES is pivotal in the pathogenesis of GvHD. Maraviroc (MVC; Selzentry®, Pfizer) is the first oral CCR5 antagonist in clinical use. The antiviral properties of MVC in HIV infection are known, but its effects on chemotaxis and immune function in patients without HIV infection have not been explored. We hypothesized that CCR5 inhibition early after allogeneic SCT would reduce lymphocyte chemotaxis and result in low rates of acute GvHD without impairing engraftment or antitumor activity.In vitro, MVC effectively and specifically inhibited CCR5 internalization and reduced RANTES-induced chemotaxis in concentrations achievable in humans, recapitulating a defect observed in homozygotes for the del32-CCR5 polymorphism. MVC had no effect on hematopoietic colony formation, T-cell mediated cytotoxicity and T-cell proliferation.Between May 2009 and March 2011, we enrolled 38 pts in a phase I/II study of reduced intensity conditioned (RIC) allogeneic SCT. Patients had high-risk features by age (median=62, range 21–74), donor source (matched related 34%, matched unrelated 50%, single-antigen mismatch 16%) and comorbidities (comorbidity index: low 55%, intermediate 34%, high 11%). Underlying diseases were AML (15), MDS (6), NHL (8), myelofibrosis (4), aplastic anemia, myeloma, CLL, Hodgkin, CML (1 each). Pts received fludarabine 120mg/m2 and IV busulfan 6.4 mg/kg followed by peripheral blood stem cells. In addition to standard GvHD prophylaxis with tacrolimus and methotrexate, MVC was given from day −2 to +30. Pharmacokinetic analysis on the first 13 pts identified 300 mg bid as the appropriate dose (Reshef, ASH 2010). MVC was well tolerated, and adverse events were similar to the expected toxicity observed in patients undergoing RIC SCT. The median time to ANC>500/μL was 15 d (range 10–27) and to platelets>20k/μL was 19 d (range 9–84). The median whole blood and T-cell donor chimerism at day 100 was 96.5% (range 0–100%) and 85% (range 0–100%) respectively.Median follow-up was 200 days (range 12–760). Among 35 evaluable patients, the cumulative incidences of any acute GvHD and grade III–IV acute GvHD at day 100 were 14.7 ± 6.2% and 2.9 ± 2.9%, respectively. Importantly, in the first 100 days, there were no cases of acute GvHD involving the liver or gut. At day 180, the rate of acute GvHD was 20.7 ± 7.1%, largely confined to the skin with low rates of GvHD in the liver (3 ± 3%) and gut (7.4 ± 5.3%). In evaluable pts who received a graft from their HLA-matched sibling (11), there was no GvHD before day 100 and only two cases of acute GvHD before day 180.We compared these results to a cohort of 38 well-matched consecutive patients treated at our institution with RIC SCT using an identical regimen but without MVC between 2009 and 2011. We observed a similar incidence of acute GvHD (all grades) at day 100 (14.7 ± 6.2 vs. 16 ± 6.1%; P=0.88), but a 64% decrease in the MVC group at day 180 (20.7 ± 7.1 vs. 45.4 ± 9%; P=0.03). The incidence rates of severe GvHD (grade III–IV) were 2.9 ± 2.9% in the MVC group vs. 5.5 ± 3.8% in the comparator group at day 100 (P=0.59) and 6.5 ± 4.5% vs. 18.1 ± 6.8% at day 180 (P=0.15).Treatment-related mortality in pts receiving MVC was low. At 1 year, non-relapse mortality rate was 7.6 ± 5.5% (control group: 15.7 ± 6.6%; P=0.35). Infectious complications were seen at a rate that is expected with RIC SCT. Recovery of lymphocyte counts and lymphocyte subsets was not impaired by MVC.We evaluated whether a protective effect against GvHD was associated with an increase in relapse. In the MVC group, the incidence of relapse was 34.2 ± 8.8% at day 180; this was not significantly different from the comparator group (43.9 ± 8.8%, P=0.44), implying preservation of the graft-versus-tumor effect with MVC. Rates of overall survival and relapse-free survival were similar in both groups.Pharmacodynamic testing revealed that sera from patients taking MVC prevented CCR5 internalization by RANTES and blocked T-cell chemotaxis in vitro, providing evidence for in vivo biological activity and supporting the hypothesized mechanism of action.In summary, inhibition of lymphocyte trafficking is a novel, specific and potentially effective strategy to reduce the incidence of acute GvHD. Disclosures:Off Label Use: Use of maraviroc in GvHD prophylaxis will be discussed. Frey:Pfizer: Speakers Bureau. Vonderheide:Pfizer: Research Funding. Porter:Pfizer: Research Funding.

CD34+ Cell Dose and Full Donor Chimerism at Day +100 Is Associated with Survival in Patients Who Received Reduced-Intensity Conditioning (RIC) With Fludarabine/Melphalan Prior To Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for Hematologic Malignancies

Nonmyeloablative, reduced-intensity conditioning extends allogeneic HSCT as a therapeutic option for elderly or frail patients with relapsed, refractory, or other high-risk hematologic malignancies. We reviewed the medical records of the first 50 patients (34 M, 16 F) at our institution who underwent fludarabine/melphalan RIC from 9/00 to 9/07 to determine factors associated with survival. The median age at transplant was 53.5 years (range 20–67 years). Most patients received HSCT for myeloid malignancies (18 AML, 11 MDS, 8 NHL, 7 MM, 2 each CML and CLL, and 1 each AMM and HL), were in remission at transplant (16 in CR, 9 in PR, 13 relapsed/refractory, and 12 untreated), received stem cells from a matched related versus unrelated donor (37 vs. 13 pts), and were a full 6/6 for sibling donors or 10/10 antigen match for URD (46 versus 4 patients with a single antigen mismatch). Half (25 pts) had received prior HSCT, and 3 had prior chemotherapy for solid tumors. 28% of patients experienced grade 3–4 acute GVHD, and 46% experienced chronic GVHD (38% extensive). 26 of 50 pts have died, 5 from relapsed disease. Only one of the deaths before day +100 was due to relapsed disease, and treatment-related mortality (TRM) at day +100 was 26% (13 pts). 24 patients (48%) are alive after a median of 24 months of follow up. Factors associated with survival on univariate analysis included CD34+ cell dose greater than the median of 5.5 × 106/L (p = 0.0463), day +100 disease remission status, (p<0.0001), day +100 chimerism status (p<0.0001), any chronic GVHD (p = 0.0005), and favorable/standard versus poor risk karyotype for myeloid malignancies (p = 0.0149). On multivariate analysis, CD34 cell dose greater than 5.5 × 106/L (RR 0.19, 95% CI 0.0389 – 0.7222, p = 0.0136) and full donor chimerism at day +100 (RR 0.05, 95% CI 0.0099 – 0.2327, p = 0.0002) remained an independent prognostic factors. Achievement of full donor chimerism was not related to CD34 cell dose (p = 0.46). In our series, achievement of full donor chimerism at day +100 is associated with 70% 2 year survival, a favorable outcome in this high risk group of patients. While the infused CD34 cell dose can occasionally be a modifiable variable, whether any other host and/or graft factors could be modified to promote the establishment of full chimerism and improve survival remains largely unknown and merits confirmation in larger series.

A Comparison of Stem Cell Source in Recipients of T-Cell Depleted Myeloablative Transplants for Leukaemia: No Difference in Mortality Using BM or PBSC.

The use of GSCF-mobilised Peripheral Blood Stem Cells (PBSC) for unrelated donor (UD) transplantation has increased dramatically since 2000. The association of PBSC with more rapid engraftment and with an increase in chronic Graft versus Host Disease (GvHD), compared to bone marrow (BM) has been reported in a number of studies. More recently the use of PBSC has been associated with an increase in transplant related mortality (TRM) and decrease in survival (OS) in T-cell replete transplants. We sought to analyse the impact of PBSC compared to BM in a cohort of UD transplant recipients, where T-cell depleting agents (in-vivo campath in >90%) were included in the transplant conditioning. The study included 145 patients transplanted between January 2000 and March 2006: CML- 35 in 1CP; acute leukaemia (AML in 61, ALL in 49)-110 in CR1 or 2. All had myeloablative conditioning regimens and received grafts with 9–10/10 matched HLA alleles. 86 patients received BM and 59 PBSC. There were no associations between the stem cell source and any transplant variable (including disease and stage). There was a trend to an increased use of PBSC in patients with a single antigen mismatch (p=0.052). All evaluable patients achieved neutrophil engraftment, with a significantly faster time to engraft in recipients of PBSC compared to BM (16 vs 20 days; p=0.0003). The incidence of acute GvHD was 46% (grade I in 50%, II in 41%, III in 8%, IV in 2%). This was significantly higher in recipients of PBSC (60%) compared to BM (36%; p=0.006), however there was no increase in either II/IV (p=0.69) or III/IV (p=0.18) disease in PBSC recipients. In univariate analysis, the presence of a single HLA mismatch (p=0.026) was the only other variable to be associated with an increase in acute GvHD. In a logistic regression model including both these variables, the use of PBSC remained significantly associated with an increase in aGvHD (OR=2.3; 95% CI 1.1,4.7;p=0.020). The TRM was 14%, 27% and 39% at 100 days, 1 and 5 years respectively. At none of these time points was the stem cell source associated with a significant difference in TRM. The 5-year incidence of chronic GvHD was 58% (BM 55%, PBSC 60%; NS), extensive disease in one third, and of relapse was 61% (BM 60%, PBSC 62%; NS). The 5-years OS was 41% with a median follow-up of 3.4 years (0.5–7.1). This was 44% using PBSC and 40% using BM (NS). In conclusion, although we observed an increase in acute GVHD with PBSC this was only of grade 1 disease. We found no association between the use of PBSC and an increased risk of chronic GVHD or of a worse transplant outcome, when compared to BM, in recipients of T-cell depleted myeloablative transplants for leukaemia.

Chronic Graft Versus Host Disease and Pretransplant Disease Status Predict Outcomes in Patients with Hematologic Malignancies Undergoing Reduced Intensity Allogeneic Stem Cell Transplantation.

Reduced intensity stem cell transplantation (SCT) is an effective treatment modality for patients with hematologic malignancies who are not candidates for conventional myeloablative SCT. We conducted a retrospective review of all patients with hematologic malignancies receiving a reduced intensity allogeneic SCT from July 2002 to July 2007. Data pertaining to patient demographics, engraftment, disease status pre and post transplant, graft versus host disease (GVHD), and HLA matching was analyzed to identify factors significantly affecting the clinical outcome. Seventy three patients, with a median age of 55 (range of 19–70) and with the diagnoses of ALL (n=8), AML (n=30), CLL (n=3), CML (n=1), Hodgkin's (n=7), non-Hodgkin's (n=7), MDS (n=11), and MM (n=6) underwent a reduced intensity SCT using a fludarabine based conditioning regimen. Thirty nine (53%) received unrelated donor grafts and 34 (47%) received sibling donor grafts. Fifty six patients (77%) received fully matched grafts whereas 17 patients (23%) had an antigen or allele mismatch. Acute GVHD grade II-IV was observed in 27 of the 73 patients and chronic GVHD was seen in 18 of the 48 patients who could be evaluated. Seventeen patients developed transplant related fatal complications and 30 patients died from disease progression or relapse. Median time to neutrophil recovery was 15 days (range of 9–41 days) and median time to platelet recovery was 18 days (range of 9–42 days). Graft failure was observed in 6 of the 73 patients. Median overall survival and disease free survival for all patients was 7.7 and 6.6 months respectively. Median overall survival for patients with persistent disease or in remission at the time of the SCT was 5.6 and 21.8 months (p= 0.01) while that for disease free survival was 5.7 and 8.4 months (p=0.06). Median overall survival with and without chronic GVHD was 25.6 and 9.4 months (p <0.0001) while median disease free survival was 18.2 and 6.0 months (p< 0.0001). Patients with limited chronic GVHD have not yet reached median overall survival while the median disease free survival was 18.4 months. Those with no or extensive chronic GVHD had medians of 9.4 and 9.2 months for overall survival and 6.0 and 9.2 months for disease free survival (p= 0.004 and p=0.02). The source of the stem cells as well as the administration of allele or single antigen mismatch grafts did not affect the outcome. Reduced intensity SCT is an effective treatment modality in patients with hematologic malignancies, though it is most effective in patients who are in remission at the time of transplant and should be offered in this setting. Patients with limited chronic GVHD had a better outcome suggesting the presence of potent anti-tumor activity of the donor immune competent cells without the detrimental effects in clinical outcome caused by extensive chronic GVHD.

Advances in HLA: Practical Implications for Selecting Adult Donors and Cord Blood Units

HLA plays a critical role in hematopoietic cell ransplantation, but a full understanding of this role is onstantly in flux because of changing technologies nd burgeoning amounts of clinical transplantation esults. Today the clinician considering unrelated donor ransplantation must also consider stem cell source: an dult donor or an umbilical cord blood unit (CBU). In he context of cord blood, the task is further compliated by the strong interaction of cell dose. Each individual carries from 10 to 12 genes that ncode the HLA-A, HLA-B, HLA-C, HLA-DR, HLAQ, and HLA-DP molecules. Most of these genes are ighly polymorphic, ranging from 13 (HLA-DRB4) to 99 (HLA-B) alleles at a locus [1]. Most of the allelic ifferences result in changes in the HLA protein seuences, thus producing potential targets for allorecogition [2]. The frequency of alleles differs in different opulations: some are common in most populations, nd others are found predominantly in only 1 or a few opulations. The same is true for HLA haplotypes—the ollection of multiple HLA alleles within a short reion of chromosome 6—which are typically inherited s a single block of genetic information. Table 1 proides examples. DNA-based testing methods provide an accurate nd powerful approach to identify HLA diversity, and hese are replacing serologic testing [3,4]. DNA testng detects specific nucleotide differences that distinuish alleles or sets of alleles. The ability to distinuish among particular alleles at a locus (ie, the esolution) depends on the methodology, the set of ligonucleotide reagents used in the assay, or both. he choice of assay depends on the purpose of the yping, eg, initial testing of newly recruited volunteers r CBUs for a registry (low to intermediate resoluion) compared with testing of transplantation candiates and a few selected potential donors or CBUs high or allele-level resolution). C

Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation.

Successful engraftment of hematopoietic stem cells from unrelated donors is influenced by disparities between the donor and recipient for HLA-A, B, and C alleles. Disparities between HLA sequence polymorphisms that are serologically detectable are termed antigen mismatches, whereas those that can be identified only by DNA-based typing methods are termed allele mismatches. Whether both kinds of polymorphisms are important in transplantation is not known. We tested the hypothesis that allele mismatches that are detectable only at the DNA level are less immunogenic than those that are serologically detectable and thereby are associated with a lower risk of graft failure after hematopoietic-cell transplantation We used DNA sequencing to define the HLA-A, B, and C alleles in 471 patients who received bone marrow from unrelated donors for the treatment of chronic myeloid leukemia after myeloablative conditioning therapy. The odds ratios for graft failure were determined for recipients of transplants from donors with a single class I allele mismatch, a single class I antigen mismatch, or two or more class I mismatches, as compared with those with no mismatch A single HLA allele mismatch did not increase the risk of graft failure, whereas a single antigen mismatch significantly increased the risk. The risk was also increased if the recipient was HLA homozygous at the mismatched class I locus or if the donor had two or more class I mismatches HLA class I antigen mismatches that are serologically detectable confer an enhanced risk of graft failure after hematopoietic-cell transplantation. Transplants from donors with a single class I allele mismatch that is not serologically detectable may be used without an increased risk of graft failure.

The Probability of Finding a Suitable Related Donor for Bone Marrow Transplantation in Extended Families

In bone marrow transplantation, the advantages of family donors over unrelated donors are threefold. (1) Family donors are better matched because they share complete haplotypes. (2) The time between the start of the search and the actual transplantation can be much shorter than for unrelated donors. (3) Related bone marrow transplantation is cheaper. We developed a procedure for calculating the probability of finding a suitable donor among cousins and blood-related aunts and uncles (the extended family). The procedure calculates the following probabilities (P): (1) P that any blood-related uncle or aunt is a suitable donor, (2) P that a suitable donor exists in all blood-related uncles/aunts (from [1]), (3) P that any cousin is a suitable donor (as in [1]), (4) P that a suitable donor exists in all cousins (from [3]), (5) P that a suitable donor exists in the entire extended family (from [2] and [4]). Additionally, we discuss the suitability of this procedure in the daily practice of donor procurement. The procedure is also suitable to search for family donors who have a single antigen mismatch with the patient. We also discuss the differences between our method and the one recently described by Kaufman (Bone Marrow Transplant 15:279, 1995).