Mixed T-cell Chimerism Research Articles

Posttransplant MRD and T-cell chimerism status predict outcomes in patients who received allografts for AML/MDS.

Allogeneic stem-cell transplant allows for the delivery of curative graft-versus-leukemia (GVL) in patients with acute myeloid leukemia/myelodysplasia (AML/MDS). Surveillance of T-cell chimerism, measurable residual disease (MRD) and blast HLA-DR expression may inform whether GVL effectiveness is reduced. We report here the prognostic impact of these biomarkers in patients allografted for AML/MDS. One hundred eighty-seven patients from FIGARO, a randomized trial of reduced-intensity conditioning regimens in AML/MDS, were alive and relapse-free at the first MRD time-point and provided monitoring samples for flow cytometric MRD and T-cell chimerism, requested to month+12. Twenty-nine (15.5%) patients had at least 1 MRD-positive result posttransplant. MRD-positivity was associated with reduced overall survival (OS) (hazard ratio [HR], 2.18; P= .0028) as a time-varying Cox variable and remained significant irrespective of pretransplant MRD status in multivariate analyses (P< .001). Ninety-four patients had sequential MRD with T-cell chimerism results at months+3/+6. Patients with full donor T-cell chimerism (FDTC) had an improved OS as compared with patients with mixed donor T-cell chimerism (MDTC) (adjusted HR=0.4; P=.0019). In patients with MDTC (month+3 or +6), MRD-positivity was associated with a decreased 2-year OS (34.3%) vs MRD-negativity (71.4%) (P= .001). In contrast, in the group with FDTC, MRD was infrequent and did not affect the outcome. Among patients with posttransplant MRD-positivity, decreased HLA-DR expression on blasts significantly reduced OS, supporting this as a mechanism for GVL escape. In conclusion, posttransplant MRD is an important predictor of the outcome in patients allografted for AML/MDS and is most informative when combined with T-cell chimerism results, underlining the importance of a GVL effect in AML/MDS.

Frequency and Clinical Significance of Mixed T-Cell Chimerism Following Myeloablative Busulfan and Cyclophosphamide Conditioning in HLA -Matched Donor Transplants for Myeloid Malignancies: An Analysis of 179 Patients Transplanted at a Single Center

Myeloablative busulfan and cyclophosphamide (BuCy2) is one of the most used preparative regimens for allogeneic hematopoietic cell transplantation for myeloid malignancies from HLA-identical (MRD) and 8 of 8 HLA-A, B, C and DRB1 allele matched unrelated (MUD) donors. However, mixed T-cell chimerism may be relatively common during the early post-engraftment phase using this regimen. The significance of early incomplete donor T-cell chimerism following BuCy2 allotransplants with respect to post-transplant outcomes is unclear. Methods: We analyzed 179 consecutive patients who underwent allotransplants for myeloid malignancies using myeloablative BuCY2 conditioning at our center. Cyclophosphamide was administered at 60mg/m2/d x 2 and busulfan was administered over four days with pharmacokinetically targeted exposure (usually 4.9 mgxhr/L) based on test dosing. Tacrolimus/methotrexate was used for GVHD prophylaxis. Uniform institutional supportive care protocols were employed for all patients. Lineage specific chimerism on T-cell (CD3) and myeloid (CD33) peripheral blood (PB) cells separated using immunomagnetic beads was performed monthly through d 100 following transplant. Patients with < 50% donor CD3 chimerism on d30 were scheduled to receive low dose DLI if available (starting at 5-10 x 10e5 CD3 cells /kg) while maintaining therapeutic tacrolimus levels. No intervention was performed if d30 donor CD3 chimerism was > 50%. Outcomes were analyzed using data extracted from our institutional database where information had been prospectively entered. GVHD diagnosis and grading were determined by a single dedicated practitioner. Results: Patient characteristics were: median age 50 (18-66), male 51%; diagnosis AML (65%), CML (23%) MDS/MPS (12%); MRD (52%) MUD (48%); graft source- PBSC 81%, BM18%, both 1%; DRI - low 12%, intermediate 57%, high 25%, very high 6%; donor CMV pos 51%, neg 49%; year of transplant- 2004-2012 (35%), 2013-2016 (32%) 2017-2021(33%). Median follow-up for surviving patients was 84 months (6-213). Kaplan-Meier (K-M) estimates for survival and relapse-free survival at 5 years for all patients were 66% (95%CI 58-73%) and 59% (51-66%). Median (range) donor chimerism on d 30 for CD3 cells was 80% (6-100%) and for CD33 cells was 100% (84-100%). Median CD3 chimerism on d 60, d 100 and d 180 were 86%(7-100%), 91% (8-100%) and 95% (12-100%) respectively. Twenty-four patients (13%) had CD3 <50% on d30 of which 9 received DLI. On multiple regression modeling (Table1) the following factors were associated with higher d 30 CD3 chimerism - diagnosis (AML vs other) (difference 0.104, p=0.001), DRI (high/v.high vs low/intermediate)(diff 0.070, p=0.03) and graft CD3 dose (>15.5x 10e7/kg vs < 15.5) (diff 0.105, p<0.001) For patients who had > 80% vs <80% donor CD3 chimerism on d 30, 5-year K-M estimates of survival and RFS were 56% vs 76% (p=0.003) and 54% vs 64% (p=0.16). Five-year cumulative incidences of NRM and relapse were 20% vs 8% (p=0.010) and 26% vs 28% (p=0.85). Cumulative incidences of grade 2-4 and grade 3-4 acute GVHD at 6 months were 49% vs 27% (p=0.002) and 20% vs 4% (p=0.001) respectively. Cumulative incidences of moderate to severe and severe chronic GVHD at 3 years were 81% vs 64% (p=0.017) and 53% vs 34% (p=0.008). On a Cox proportional-hazards model adjusting for significant patient, disease and transplant covariates, d 30 CD3 chimerism > 80% was associated with a significantly higher risk of NRM (HR 3.42, p=0.007), aGVHD gd 2-4(HR1.79, p=0.013), aGVHD gd 3-4 (HR 2.66, p=0.008) and severe cGVHD (HR 1.84, p=0.011). However, d 30 CD3 chimerism was not significantly associated with relapse, survival and relapse-free survival (Fig 1). Conclusions: These data suggest that while pharmacokinetically-targeted BuCy2 as administered is almost always myeloablative (CD33 chimerism 100%), early mixed T-cell chimerism is relatively common (CD3 chimerism < 80% in 50% of patients). d30 CD3 chimerism ≥80% is not associated with lower relapse rates or better survival outcomes, but is associated with higher rates of acute and chronic GVHD. No intervention to improve CD3 chimerism appears necessary for patients with d 30 CD3 chimerism between 50 and 80%. Patients with ≥ 80% CD3 chimerism at d 30 may benefit from more aggressive measures to prevent acute and chronic GVHD Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mixed T cell CHIMERISM in bone marrow at day+30 after allogeneic stem cell transplantation: Correlation with acute GVHD grades III-IV and no impact on relapse and overall survival.

Early chimerism analysis is important to assess engraftment in allogeneic hematopoietic stem cell transplantations. We retrospectively investigated the impact of T-cell chimerism at day 30 in bone marrow on acute graft-versus-host disease (aGVHD), relapse, and overall survival in 142 adult allo-transplanted patients. The majority of patients (89%) received myeloablative conditioning and 90% have undergone T-cell replete donor graft. At day 30, 103 patients showed T-complete chimerism with prevalence in haploidentical transplants, whereas 39 cases had CD3+ mixed chimerism, including 30 patients transplanted with HLA identical donors, and 21 with T-cell donors<90%. T-cell chimerism at day 30 was weakly inversely related to aGVHD grades II-IV (p=.078) with no cases of grades III-IV aGVHD in patients with CD3+ <95%. Mixed T-cell chimerism did not impact on relapse (p = .448) and five of the seven patients who relapsed had T-cell chimerism ≤90%. Older age and active disease at transplant had a statistically significant negative effect on overall survival (p = .01 and p = .0001, respectively), whereas mixed CD3+ chimerism did not. T lymphocyte chimerism analysis at day +30 in bone marrow could identify allo-transplanted patients at major risk of aGVHD grades III-IV (CD3+ donors >95%) mainly post-myeloablative conditioning regimen.

Outcomes After Donor Lymphocyte Infusion in Patients With Hematological Malignancies: Donor Characteristics Matter

In the context of T-cell depletion, failing to achieve full donor chimerism (FDC) entails higher risk of graft loss and disease relapse. Donor lymphocyte infusion (DLI) is an adoptive immunotherapy for mixed chimerism (MC) or relapsed disease after reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (HSCT). Nevertheless, little is known of factors associated with attaining FDC or disease remission. We carried out a retrospective study with 100 adult patients to identify patient and donor factors that can predict achievement of FDC and disease remission and describe complications after DLI. Indications for DLI were T-cell MC in 61 patients and relapsed disease in 39 patients. Forty patients (65.6%) with MC attained T-full donor chimerism (T-FDC), with higher responses seen in patients whose donors were female (81.5% versus 52.9%, P = .004) and cytomegalovirus negative (76.5% versus 52%, P = .004). However, only patients with younger donors (<30 years old) compared to older donors (94.4% versus 53.5%, P = .013) and those attaining unfractionated whole blood (UWB) FDC after DLI (76.6% versus 28.6%, P < .001) had a survival benefit and subsequently a better graft-versus-host disease (GvHD)–free/relapse-free survival. Nineteen of 39 patients (48.7%) with relapsed disease achieved remission after DLI. In this cohort, attaining T-FDC impacted favorably in disease control (76.7% versus 12.5%, P = .012) and improved survival (45.5% versus 12.5%, P = .007). In the whole population, the cumulative incidence of acute GvHD (aGvHD) at day 100 after DLI was 23%, and chronic GvHD (cGvHD) at 1 year after DLI was 22%. In the whole population, donor age was also a determining factor for aGvHD, because patients with younger donors had a lower incidence of aGvHD (8% versus 36%, P = .021). The cGvHD was more likely to occur in patients who converted to T-FDC (34% versus 10.3%, P = .025). Donor characteristics are increasingly considered when deciding approaches for HSCT. Donor age should be considered when planning HSCT, as well as doses and scheduling of DLI. As per our experience, this should be done alongside T/UWB chimerism to achieve the maximal clinical benefit with less associated toxicity. Selection of younger male donors from stem cell registries can minimize the risk of GvHD and improve survival.

Chimerism of Blood T-Cells and Leukemia Lineage Cells Following Myeloablative Hematopoietic Cell Transplantation Is a Risk Factor for Relapse and Chronic Graft-Versus-Host Disease

Background: Mixed chimerism of blood leukemia lineage cells has been reported to be highly predictive of relapse (Mattsson J et al. Leukemia 2001), but the utility of T-cell chimerism is controversial.Methods: Chimerism of T-cells (CD3 +) and leukemic lineage cells (CD13 +/CD33 + for myeloid malignancies and CD19 + for B-lymphoid malignancies) was measured in the peripheral blood for 600 hematopoietic cell transplant (HCT) recipients at 3-months post-transplant. Conditioning was myeloablative (fludarabine+busulfan+4GyTBI) and GVHD prophylaxis was with ATG+CsA+MTX.Results: Mixed (<95% donor) chimerism was present in 6% of patients in the leukemic lineage and 16% of patients in T-cells. Compared to patients with complete chimerism (≥95%), mixed chimerism predicted a significantly greater incidence of relapse (52% vs. 27%, P=0.044), and surprisingly, also a greater incidence of cGVHD (43% vs. 23%, P=0.028). Patients with mixed leukemic lineage chimerism also had poorer cGRFS (7% vs. 39%, P<0.001) and OS (29% vs. 55%, P<0.001). Mixed T-cell chimerism predicted a significantly greater incidence of relapse (46% vs. 24%, P<0.001) and lower cGVHD incidence (9% vs. 31%), with no difference in cGRFS or OS. The sensitivity/specificity/positive predictive value (PPV)/negative predictive value (NPV) of mixed leukemic lineage and T-cell chimerism for relapse were 65%/55%/35%/81% and 66%/57%/60%/79%, respectively. Sensitivity/specificity/PPV/NPV was similarly poor for cGVHD. In patients with complete leukemic lineage chimerism at 3-months, a ≥5% drop in donor chimerism at any future timepoint had sensitivity/specificity/PPV/NPV of 82%/79%/79%/82% for subsequent relapse.Conclusion: In the setting of myeloablative conditioning and ATG-based GVHD prophylaxis, mixed chimerism at 3-months post-transplant is a risk factor for subsequent relapse. However, the utility of these measurements in guiding medical interventions may be limited by insufficient predictive values. Nevertheless, patients with mixed chimerism may be candidates for more intensive surveillance. DisclosuresStorek: Atara Biotherapeutics: Other: Site PI, Research Funding.

Long-term survival with mixed chimerism in patients with AML and MDS transplanted after conditioning with targeted busulfan, fludarabine, and thymoglobulin.

We evaluated long-term outcome in 40 patients with MDS or AML, transplanted from related or unrelated donors following conditioning with targeted busulfan (Bu, over 4 days), fludarabine (Flu, 120 [n = 23] or 250 [n = 17] mg/m2) and thymoglobulin (THY). Compared to 95 patients conditioned with Bu/Cyclophosphamide (Cy) without THY, BuFluTHY-conditioned patients had lower rates of chronic graft-vs.-host disease (GVHD). Adjusted hazard ratios (HR) for BuFlu(120)THY and BuFlu(250)THY-conditioned patients were 1.60 (95% confidence interval (CI) 0.66-3.86) and 1.87 (0.68-5.11), respectively, for relapse; 0.77 (0.30-1.99) and 1.32 (0.54-3.23) for non-relapse mortality; 0.81 (0.42-1.57) and 1.38 (0.72-2.57) for overall mortality; and 0.78 (0.30-2.05) and 1.62 (0.63-4.41) for relapse or death (failure for relapse-free survival). At one year, 45% of BuFlu(120 or 250)THY-conditioned patients had mixed CD3+ chimerism compared to 0% with BuCy (p < 0.0001). None of 7 patients with long-term mixed chimerism had chronic GVHD; two relapsed, five remained stable mixed chimeras. THY is effective in reducing chronic GVHD, and long-term mixed T-cell chimerism can be compatible with relapse-free survival. However, Thy may also be associated with an increased risk of relapse and, dose-dependent, with non-relapse mortality.

Myeloid Mixed Chimerism and Relapse in Myelofibrosis after Allogeneic Stem Cell Transplant

Background:Allogeneic stem cell transplant (SCT) remains the only potential curative treatment for myelofibrosis (MF). However, relapse after transplant is one of the leading causes of death. The role of mixed myeloid chimerism (MMC) in predicting post-transplant molecular and morphologic relapse is not well studied.Methods: Using a large single-center database, all consecutive patients with primary or secondary MF who underwent first allogeneic SCT in the period 2005-2015 were included in the study. Patients were defined to have MMC if they were found to have less than 95% of donor myeloid cells at any point over the duration of their follow up. Peripheral blood chimerism analysis was performed using 8 highly polymorphic microsatellite markers (Integrated DNA Technologies, Coralville, IA) in a multiplex PCR assay. The primary end point was to assess the correlation of MMC with any evidence of morphologic and/or molecular relapse.Results: A total of 85 patients (47 males, 38 females) were identified with a median age at transplant of 57 years (27-74). Among patients with molecular data (n=80), 60% had positive pre-transplant molecular markers. A total of 36 (43%) patients with full chimerism data (n=83) had MMC. All patients with MMC developed morphologic and/or molecular relapse, except for two; the first patient converted to full donor chimerism after immunosuppression reduction and the second patient had progressive myeloid chimerism and graft failure. Among patients with positive molecular testing and available chimerism data (n=46), 22 patients had MMC, of which 95% (n=21) had molecular relapse. Similarly, for patients with available chimerism data and negative pre-transplant molecular testing (n=32), 93% (n=14) of the 15 patients with MMC experienced eventual morphologic relapse. On the other hand, none of the patients with full donor myeloid chimerism (n=47) progressed during the study period, except for one patient with mixed T-Cell chimerism. With a median follow up of 49 months, the 5-year PFS was 7% for those with MMC vs 62% for patients with full donor myeloid chimerism (p<0.0001).Conclusion: Given the high sensitivity of chimera PCR DNA testing and its strong association with molecular and morphologic relapse, we speculate that myeloid chimerism testing, alone or combined with clonal molecular testing, would serve as one of the earliest and best markers to predict MRD and relapse in myelofibrosis post-transplant. DisclosuresOran:Astex: Research Funding; AROG: Research Funding; Celgene: Research Funding. Bose:Incyte Corporation: Honoraria. Pemmaraju:LFB: Consultancy, Honoraria; stemline: Consultancy, Honoraria, Research Funding; novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria; cellectis: Research Funding; roche diagnostics: Consultancy, Honoraria; affymetrix: Research Funding; abbvie: Research Funding. Verstovsek:Galena BioPharma: Research Funding; Blueprint Medicines Corp: Research Funding; CTI BioPharma Corp: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Gilead: Research Funding; Astrazeneca: Research Funding; Roche: Research Funding; Bristol Myers Squibb: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Lilly Oncology: Research Funding; Pfizer: Research Funding; Promedior: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Astrazeneca: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Research Funding; Blueprint Medicines Corp: Research Funding; Genentech: Research Funding; Promedior: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Lilly Oncology: Research Funding.

Host T-Cells Contribute to Immune Reconstitution and Improved Outcome in Recipients of CD34 Selected T-Cell Depleted Allografts (TCD HCT) with Full Hematopoietic Cell but Persistent Mixed T-Cell Chimerism

<h3>Background</h3> Isolated mixed T-cell chimerism in otherwise fully engrafted recipients of TCDHCT has been associated, in some series, with risk of graft rejection or relapses and an indication for DLI. Thereto, we have analyzed the impact of donor chimerism on outcomes in a prospective clinical trial of conditioning regimens used with CD34+ selected TCDHCT. <h3>Methods</h3> The trial (NCT0119066) compares A) HFTBI, Thiotepa and cyclophosphamide with B) Busulfex, melphalan and fludarabine or C) Clofarabine, melphalan and thiotepa as conditioning for CD34+ TCDHCT used to treat high risk hematologic malignancies. As part of the trial, we sequentially assessed lineage-specific donor chimerism in the marrow and the myeloid cells and T-cells in blood by analysis of donor and host-specific STRs in cell fractions isolated by immunotype. In the landmark analysis, quartiles of donor chimerism were compared for T-cell levels, and clinical outcomes. <h3>Results</h3> 283 consecutive patients, transplanted between 5/13/2010 and 12/31/2016 (106 in Arm A, 144 in Arm B, 33 in Arm C). All engrafted by day 10-11. While 3 pts in Arm B and 1 in C had late graft failure, all others from 3 mos on had full donor chimerism in marrow and myeloid blood cells. However, at 3 mos median donor CD3⁺ T-cell chimerism (MDTC), was mixed in each arm but significantly lower in Arm B (10%) vs A (50%) and C (63%) (p<0.001). While MDTC for Arms A and C rose to 80% and 85% by 6 mos and ≥95% by 1yr, but in Arm B was 25% at 6 mos, 55% at 1 yr and 65% at 2 yrs. To assess the impact of this sustained mixed chimerism, we did a landmark analysis at 3 months, comparing subsequent T-cell recovery and clinical outcomes for pts in the first (lowest) and fourth (highest) quartile of T-cell chimerism in Arms A and B. As shown in Table 1, CD3⁺ and CD8⁺ T-cells in both arms were significantly higher in Q1, as were CD3⁺CD4⁺ cells in Arm B. Non-relapse mortality (NRM) at 2 yrs was also significantly lower for Q1 pts in both arms but relapse risks were not. In Arm B, but not A, 2yr OS and DFS were also significantly better in Q1 vs Q4. <h3>Conclusions</h3> Conditioning with Bu/Mel/Flu (Arm B) secured durable engraftment and hematopoietic reconstitution but a high proportion of pts have isolated and persistent mixed T-cell chimerism. However, surviving host T-cells can expand, persist and contribute significantly to earlier T-cell recovery and long-term reduction of NRM without increasing risk of graft rejection, relapse or need for DLI.

Early mixed T-cell chimerism is predictive of pediatric AML or MDS relapse after hematopoietic stem cell transplant.

Patients with acute myeloid leukemia (AML) who relapse after hematopoietic stem cell transplantation (HCT) have dismal outcomes. Our ability to predict those at risk for relapse is limited. We examined chimerism trends post-HCT in 63 children who underwent HCT for AML or myelodysplastic syndrome (MDS). Mixed T-cell chimerism at engraftment and absence of chronic graft versus host disease (cGVHD) were associated with relapse (P = 0.04 and P = 0.02, respectively). Mixed T-cell chimerism at engraftment was predictive in patients without cGVHD (P = 0.03). Patients with engraftment mixed T-cell chimerism may warrant closer disease monitoring and consideration for early intervention.

High-Exposure, Targeted Daily Busulfan and Fludarabine-Based Conditioning for Children Undergoing Hematopoietic Stem Cell Transplantation for Inherited Metabolic Disorders: Outcomes at a Single Center

Allogeneic hematopoietic stem cell transplantation (HSCT) has emerged as standard therapy for children suffering from various life-threatening inherited metabolic disorders (IMD). Historically, successful outcomes have been limited by high rates of both graft failure and regimen-related toxicity. Real-time busulfan pharmacokinetic monitoring, combined with once daily dosing, has allowed for more precise delivery of targeted exposures during transplant conditioning. Recently, the Utrecht group reported highly favorable outcomes following a high-dose busulfan and fludarabine-based preparative regimen in patients with a variety of diagnoses, particularly in comparison to historical regimens that employ two or more alkylating agents. We report our experience using the same regimen specifically in patients with IMD. Since July 2014, 24 children undergoing first HSCT for IMD were conditioned with once daily IV busulfan (4 days; total regimen exposure of 90 mg x h/L) and once daily IV fludarabine (40 mg/m2/day x 4 days). Busulfan pharmacokinetic monitoring was performed on each of the first 3 doses (days); subsequent doses were altered to achieve the desired total regimen exposure. Serotherapy use was donor-dependent: HLA-matched related donor (MRD, n = 5), none; unrelated UCB (n = 16), thymoglobulin (2.5 mg/kg/day, days - 8 to -5); HLA-matched unrelated donor (MUD, n = 1), thymoglobulin (2.5 mg/kg/day, days - 5 -to -2); and, related HLA haploidentical marrow (n = 2), alemtuzumab (0.5 mg/kg divided days -11 and -10), +/- rituximab (375 mg/m2, day -12). All patients received CSA for GvHD prophylaxis. Additional GvH prophylaxis depended upon the allograft source: UCB, prednisolone; MRD/MUD, MMF; related haploidentical, MMF and post-transplant cyclophosphamide. Growth factor support was administered to recipients of UCB or haploidentical grafts. IMD diagnoses were as follows: Hurler syndrome (n = 10); cerebral adrenoleukodystrophy (n = 9); metachromatic leukodystrophy (n = 1); osteopetrosis (n = 1); fucosidosis (n = 1); alpha-mannosidosis (n = 1); and, beta-mannosidosis (n = 1). At a median follow-up of 229 days (range, 15 - 743), 22 of 24 patients survive, with a Kaplan Meier estimate of survival at one year of 86%. Causes of death were pulmonary bronchiolitis obliterans (BOS, n = 1) and graft failure (n = 1). Twenty-two patients had initial donor neutrophil recovery at a median day +14 (range, +11 to +21); for these patients, the median duration of severe neutropenia (ANC < 500/μL) was 7.5 days (range, 4 to 16 days). Of 20 patients who at the time of analysis are ≥ 30 days from HSCT, 18 had platelet engraftment (≥20,000/μL, transfusion unsupported) at a median day +22.5 (range, +10 to +94). One patient developed Grade II aGvHD (haploidentical marrow graft) which was successfully treated; no patients developed Grade III-IV aGvHD. One patient developed chronic GvHD with fatal BOS (UCB). Regimen-related complications were as follows: veno-occlusive disease, n = 0; hemorrhagic cystitis, n = 1; diffuse alveolar hemorrhage n = 1; idiopathic pneumonia syndrome, n = 1; immune-mediated cytopenias, n = 2; CMV viremia, n = 3; and, adenovirus infection, n = 1. Twenty patients (83%) have demonstrated durable, complete donor myeloid engraftment following the regimen. Of these, all have demonstrated mixed donor-recipient T-cell chimerism. Four patients, (all UCB grafts), have experienced graft failure: aplastic graft failure, n = 3, and autologous hematopoietic recovery, n = 1. Three of these patients underwent second HSCT and 2 are alive and engrafted while the other died of complications following second transplant. One patient awaits second transplant. In summary, this regimen of high-exposure, targeted daily busulfan and fludarabine in children with IMD provides favorable engrafted survival with minimal toxicities. DisclosuresNo relevant conflicts of interest to declare.

Immunological Profile of Patients after HSCT Using the FCC Conditioning Regimen for Treatment of Severe Aplastic Anemia Shows Sustained Mixed T-Cell Chimerism Is Due to Persistence of Recipient CD8 T Cells and Indicates Potential Basis for Tolerance and Extremely Low Incidence of Graft Versus Host Disease

Mixed T-cell chimerism is frequent and persists despite discontinuation of immunosuppression after hematopoietic stem cell transplantation (HSCT) for severe aplastic anemia (SAA) using the fludarabine, cyclophosphamide and alemtuzumab/campath (FCC) conditioning regimen. To investigate the basis for long-term co-existence of donor and patient T cells and remarkably low rates of graft versus host disease (GVHD), the lymphocyte composition and lymphocyte subset chimerism of adult patients transplanted for SAA using FCC was assessed.PatientsWe have transplanted 42 patients (pts) for acquired idiopathic SAA using FCC conditioning from 2007- March 2015 at King's College Hospital. Median age was 33 years (range 15-63). BM was stem cell source in 7(17%) pts and PBSC in 30 (83%). Median cell dose was 6.85 (1.9-12.4) CD34+ cells x106/Kg. Donor was matched sibling in 11(26%) and unrelated in 31 (74%) pts, 6 of whom also received 2 Gy TBI for 9/10 match. Post graft immunosuppression comprised ciclosporin (CSA) alone. Primary graft failure occurred in 1 pt (2%). Acute GVHD was seen in 6 (14%) pts (all grade I-II), and chronic GVHD in 5 (12%) patients (all skin, only 1 severe in a 9/10 match patient). Factors associated with cGVHD were previous aGVHD (p=0.035) and CD3+ chimerism at day+100 ³ 90% (p=0.006). Median follow-up of survivors was 41 months (4.3-96), with 93% overall survival and 90% event-free survival. At 1, 2 and 3 years post HSCT, 4/30, 20/23 and 16/16 patients, respectively, had discontinued CSA.ResultsComparison to 11 healthy age-matched individuals showed prolonged lymphopenia and abnormal proportions of T, B and NK cells. T cells were profoundly deficient comprising only 1.76% of lymphocytes at day 30, rising to 43.8% by day 360 but still significantly below normal (66%, p=0.018). CD8 T cells recovered faster than CD4 T cells producing inversion of the normal CD4 to CD8 T cell ratio. Composition of na•ve, memory and effector subsets within the CD4 T cell population was near normal by day 360 and na•ve CD4 T cells expressed CD31 indicative of renewed thymopoiesis. In contrast, CD8 T-cell subset composition remained abnormal at day 360 due to a high proportion of effector T cells (57.2% of CD8 T cells compared to 9.3% for healthy individuals, p=<0.005). T-cell subsets from five patients were isolated by fluorescence-activated cell sorting, and chimerism determined by analysis of informative alleles from polymorphic short tandem repeat loci. Results shown in the figure below revealed that mixed T-cell chimerism at day 360 was principally due to persistence of patient CD8 T cells, with notable contribution of the effector subset. Follow-up analysis of three patients at >2 years after HSCT showed the mixed chimerism profile within T-cell subsets was stable. A significant correlation was observed between lower donor T-cell chimerism at day 360 and CMV reactivation or EBV viremia early after HSCT (p=0.036), suggesting that antigen-driven expansion of virus-specific patient CD8 effector T cells may substantially contribute to persistent mixed T-cell chimerism.Sustained co-existence of donor and patient T cells and low rates of GVHD indicates conditions favor mutual tolerance. Findings suggested a potential role for cells with immunomodulatory properties. B cell recovery was robust, with normal numbers by day 100. Notably, the B cell population present early after HSCT contained a significantly increased proportion of cells with an immature transitional phenotype (CD24+ CD38+, CD27- IgMhigh IgDhigh 17.1% compared to 2.7% for healthy individuals, p=<0.005) and significantly more B cells produced IL-10 after in vitro stimulation with CD40L (14.6% compared to 6.3% for healthy individuals, p=0.013), consistent with the known immunosuppressive activity of immature B cells. Furthermore, proportions of regulatory T cells (CD4+ CD25high CD27+ FoxP3+) within the CD4 T-cell population were normal (range 4.2-7.4% compared to 5.3% in healthy controls).ConclusionsPatient-derived effector CD8 T cells shape mixed T-cell chimerism after FCC conditioning and presence of regulatory B and T cells may contribute to long-term tolerance of mixed T-cell chimerism with associated low incidence of GVHD in the absence of post graft immunosuppression.Note: Marsh JCWand Barber LD joint last authors [Display omitted] DisclosuresKulasekararaj:Alexion: Consultancy.

Lineage Specific Chimerism Analysis Reveals That Mixed Donor T-Cell Chimerism Is Common in the Early Post-Transplant Period Following Myeloablative Allotransplants from HLA-Matched but Not HLA-Haploidentical Donors: A Multivariable Analysis of Allografted Patients from a Single Center

Failure to achieve complete donor T-cell chimerism in the early post-transplant period may impair the graft-versus malignancy effect and contribute to a higher risk of disease relapse. Lineage specific chimerism analysis (LSCA) of T-lymphocytes versus myeloid cells is routinely performed following reduced-intensity or non-myeloablative hematopoietic cell allografts. However, many centers do not perform LSCA following myeloablative conditioning. We performed LSCA in all consecutive patients receiving a first T-replete allotransplant using myeloablative conditioning as defined by the CIBMTR (Giralt 2009 BBMT 15;367) between 2/2006 and 2/2015 (n=232; patient characteristics: median age 49 (18-73); Female 50%, Black 19%, Asian 3% white 78%; donor MRD 41%, MUD 39%, Haploidentical 20%; graft PBSC 88%, BM 11.5%, both 0.5%; diagnosis AML 52%, ALL 18%, MDS 11%, CML 11%, NHL 4% MPS1%, CLL1%, HL 1%; DRI low 10%, intermediate 51%, high 29%, very high 10%. Our objectives were to determine the rate of full-donor and mixed T-cell chimerism in the early post-transplant period (d 30 and d 90) , to determine the association of T-cell chimerism with patient, disease and regimen specific factors, and to determine whether early mixed chimerism impacts post-transplant outcomes following myeloablative conditioning. LSCA was performed on peripheral blood using a RoboSep instrument for automated sorting of CD3-positive T-cells and CD33 positive myeloid cells to 96.5-100% and 98.3-100% purity respectively, and short tandem repeat analysis by PCR. Full donor chimerism was defined as > 90% donor derived cells. Probability of achieving full donor T-cell chimerism in evaluable patients on d 30 and d 90 post transplant were 55% and 71%. In contrast the probabilities of achieving full-donor myeloid chimerism were 99.5% and 93.5% respectively. On univariate analysis the following factors were significantly associated with achievement of full-donor T-cell chimerism on d 30: donor type (haploidentical 100%, MRD 39%, MUD 47%, p<0.001), diagnosis (AML 58%, ALL 60%, MDS/MPS/CML 35%, NHL/HL/CLL 90%, p=0.003), conditioning regimen (busulfan based 37%, TBI based 56%, post-transplant Cy 100%, p<0.001), and on d 90: donor type (haploidentical 100%, MRD 56%, MUD 70%, p<0.001), diagnosis (AML 76%, ALL 77%, MDS/MPS/CML 49%%, NHL/HL/CLL 100%, p=0.002), conditioning regimen (busulfan based 56%, TBI based 77%, post-transplant Cy 100%, p<0.001). For multivariate analysis (Table 1) the exact logistic regression method was used to accommodate the nature of the data. The odds ratios (OR) were estimated by exponentiating median unbiased estimates of regression coefficients. Donor type (haploidentical vs other) and diagnosis (MDS/MPS vs AML) were significant factors. Our institutional approach to patients failing to achieve full-donor chimerism by d 90 is to withdraw immnosuppressive therapy in the absence of active GVHD if T-cell chimerism is > 50% or to administer DLI (starting at 1 x 10e6 CD3+ cells/kg) for patients with CD3 chimerism < 50% . Using this approach long term outcomes for patients who failed to achieve full-donor CD3 chimerism by d 30 were not significantly different from those achieving this threshold (2 yr estimated survival -81% vs 67%; disease-free survival 62%% vs 57%; 6 month CI of grade 2-4 acute GVHD 33% vs 41%; 2 yr CI of non-relapse mortality 9% vs 15%, relapse 29% vs 28%; p=NS for all). These data demonstrate that failure to induce early full-donor T-cell chimerism is relatively common following myeloablative allotransplantation using HLA-matched but not haploidentical donors. Although spontaneous improvement can occur, routine LSCA followed by immunologic manipulation of patients with suboptimal chimerism may assist in preventing adverse long term outcomes.Table 1D 30 - OR = odds ratio of achieving full-donor CD3 chimerismFactorEffectOR95% CIP valueDiagnosisALL vs AML0.980.36 - 2.651.000MDS/MPS/CML vs AML0.370.13 - 0.940.035NHL/HD/CLL vs AML5.220.48 - 2700.255DonorTypeMRD vs MUD0.630.29 - 1.350.266Haploidentical vs MUD57.312.0 - ∞<0.001Table 2OR = odds ratio of achieving full-donor CD3 chimerismFactorEffectOR95% CIP valueDiagnosisALL vs AML0.700.20 - 2.490.719MDS/MPS/CML vs AML0.150.04 - 0.48<0.001NHL/HD/CLL vs AML3.870.68 - ∞0.216Donor TypeMRD vs MUD0.350.12 - 0.980.045Haplo vs MUD19.03.64 - ∞<0.001 DisclosuresNo relevant conflicts of interest to declare.

Fludarabine, Melphalan and Alemtuzumab Conditioned Reduced Intensity (RIC) Allogeneic Hematopoietic Cell Transplantation for Adults Aged >40 Years with De Novo Acute Lymphoblastic Leukemia: A Prospective Study from the UKALL14 Trial (ISRCTN 66541317)

In the donor versus no donor analysis of the UKALL12/E2993 trial in adult acute lymphoblastic leukaemia (ALL), there was a significant improvement in overall survival (OS) and reduction in relapse in the donor arm. However the OS benefit did not extend to older patients in whom myeloablative allogeneic hematopoietic cell transplant (alloHCT) related mortality was 35% at 2 years and outweighed the reduction in relapse risk. The overall outcome of older patients in this trial was poor, with patients >40 and 50 years having 23% and 15% 5 year survival respectively. In our current UK National Cancer Research Institute UKALL14 study all patients >40 years in CR, regardless of Philadelphia (Ph+) status and other high risk factors, are considered "high risk" and recommended a reduced intensity conditioned (RIC) alloHCT with a matched sibling (sib) or 8/8 matched unrelated donor (MUD) after a 2 course induction and high dose methotrexate. The primary endpoint is event free survival (EFS). We report here the early outcome of 88 patients with at least day 100 follow up (FU) (median FU 18 months) who received RICalloHCT on trial.Conditioning was with fludarabine 30mg/m2 d -6 to -2, melphalan 140 mg/m2 d -2 and alemtuzumab 30mg d -2 to-1 (MUD) or d-1 (sib). Graft versus host disease (GVHD) prophylaxis was ciclosporin A only. Nine patients did not receive alemtuzumab as per protocol. Multilineage chimerism (MC) and minimal residual disease (MRD) were assessed 3 monthly post alloHSCT. Escalating doses of donor lymphocyte infusions (DLI) were given for T-cell mixed chimerism or MRD, starting dose 1 x 106 CD3 cells/kg, escalating by half log increments every 3 months.Of 511 registered patients, 306 were >40 years old; 127 of those have completed RIC allograft, 88 of whom have sufficient FU to report. Median age was 51.5 years (range 41 to 64). Donor was sib in 24 and MUD in 64 patients, respectively. Median WBC at diagnosis was 7 x 109/l (0.6-557). Forty one of 77 (53%) evaluable patients had high risk cytogenetics, 22 (25%) were Ph+. Twelve of 56 (21%) with MRD data were MRD +ve pre-alloHCT.Post-alloHCT, myeloid engraftment occurred in 86/88 patients at a median of 13 days, 2 had missing data. Acute graft versus host disease (GVHD) occurred at grade 1 in 30 patients (34%) and grade 2-3 in 8 patients (9%). Thirty patients developed chronic GVHD (34% of 87/88 patients surviving to D100), 13 limited and 17 extensive. Of 12 patients who suffered transplant-related mortality (TRM), 6 died of infection (one post-transplant lymphoproliferative disease), 4 of organ toxicity and 2 of GVHD. TRM was not associated with age or donor type. Sixteen patients relapsed at a median time of 317 days (range 110-1034). Of those, 9 (of 15 with data) had high risk cytogenetics (EFS, p=0.38) and 6 (of 11 with data) were MRD +ve pre-alloHCT (EFS, HR 3.35, p=0.047). Twenty seven patients in total received 67 DLI (median maximum dose 3 x 106 CD3 cells/kg): 15 for mixed chimerism, 4 for rising MRD and 8 for both. Five patients (19%) developed post-DLI GVHD at grade 1 (n=4) and grade 2 (n=1). MC data is available for 36 patients receiving alemtuzumab and is shown in figure 1 where dark grey bars represent full and light grey bars mixed chimerism, respectively. DLI are denoted by black dashes. Mixed chimerism was seen in 22 of 36 patients (61%) who received alemtuzumab and so far 26 of 36 have achieved full donor chimerism (median 9 months), 10 after DLI (38%). Figure 2 shows Kaplan Meier curves of OS 76% (64-84, 95% CI) and EFS 68% (55-77)at 18 months.This is the first prospective study of RIC alloHCT in older adults with ALL; these early results demonstrate its feasibility within a large, multicentre trial. T-cell mixed chimerism was common at 1st MC assessment but early data indicate that conversion to full donor chimerism with DLI is a safe and realistic possibility. The impact of mixed chimerism (and pre-emptive DLI) on relapse remain to be evaluated. Severe GVHD and TRM were relatively low, such that the EFS of chemotherapy then RIC alloHCT, albeit with short-term FU, may be higher than expected for chemotherapy alone in this age group. [Display omitted] [Display omitted] DisclosuresCopland:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fielding:Amgen: Consultancy, Honoraria.

King’s College Hospital FCC Conditioning for Severe Aplastic Anemia Induces Tolerance with Mixed T-Cell Chimerism and Extremely Low Incidence of Gvhd

We have previously shown that HSCT conditioning regimen for severe aplastic anemia (SAA) using fludarabine, cyclophosphamide and alemtuzumab (FCC), is associated with very low rate of GvHD (especially chronic, cGVHD), and excellent overall survival (OS) of 83% and 95% for matched unrelated (MUD) and matched sibling donor (MSD) HSCT, respectively. Sustained full donor myeloid chimerism occurred most frequently with mixed T-cell chimerism, and this persisted despite discontinuation of post graft immunosuppression with ciclosporin (CSA). We proposed that prolonged mixed T-cell chimerism with alemtuzumab conditioning may facilitate tolerance and hence a low risk of GVHD.We have investigated the basis for the mixed T-cell chimerism in a cohort of 35 patients, median age 32y (range 15-63), transplanted for acquired idiopathic SAA from 2007 - 2014 at King’s College Hospital. FCC conditioning comprised fludarabine 30 mg/m2 iv (day -7 to -4), cyclophosphamide 300 mg/m2 iv (day -7 to -4) and alemtuzumab (median dose 70mg, range 45-100). CSA was used as post graft immune suppression for 9 months, followed by dose tapering to zero over the next 3 months depending on PB chimerism. Stem cell source was BM in 7(20%) patients and 28(80%) PBSC, with a median cell dose of 6.8 (1.9-12.4) CD34+ cells x 106/Kg. Donor type was MSD in 9(26%) pts and UD in 26(74%), of whom 20 were matched (MUD) and 6 were 9/10 match ( received FCC with 2 Gy TBI). Median time to neutrophils >0.5x109/l was 12(10-20) days and to platelets >50x109/l was 10(9-61) days. Primary graft failure occurred in only one patient (2%). Acute GvHD was seen in 6(17%) pts (all grade I-II), and chronic GvHD in 5(14%) pts (3 mild, 1 moderate, 1 severe, all with only skin involvement). On univariate analysis with Cox regression model, factors significantly predicting cGVHD were previously diagnosed acute GVHD (p=0.035) and CD3+ chimerism at day+100 ≥90% (p=0.006). Four patients developed autoimmune anemia post HSCT (2 hemolytic anemia, 2 PRCA). Median follow-up was 30 months (4.3-86.8), OS 94% and event free survival 90%. One year TRM was 6%.Of 32(91%) patients evaluable for chimerism, 8 (23%) were fully donor chimeric for unfractionated BM, PB CD15+ and PB CD3+, while 24 (77%) patients had persistent mixed CD3+ chimerism. Median CD3+ chimerism values were 77% at day+30, 60% at day+60, 41% at day +100,57% at +day180, 70,5% at 1 year, 77% at 2 years and 80% at 3 years. The proportion of evaluable patients no longer taking CSA was 0/23 at 1yr, 9/19 at 1.5yr, 12/16 at 2yr, 12/12 at 2.5yr and 4/4 at 3yr. PB immunophenotyping was performed in 19 patients. During the first year post HSCT, there was profound and prolonged lymphopenia, NK cells recovered first but numbers remained below normal. T cells comprised only 2.6% of lymphocytes at day 30, rising slowly to 41% at day 360 but still significantly below normal (66% in 11 aged-matched healthy individuals), with deficiency greatest for CD4+ T cells. Despite slow recovery of CD4+ T cells, percentage of T-regulatory cells (CD4+ CD25high CD27+ FoxP3+) within this population was normal (range 4.2-7.4% for all time points compared to 5.3% for healthy individuals). Patients aged <50y showed recovery of naïve T cells that expressed markers of recent thymic emigrants (CD31+ CD62L+ CD45RA+), indicating renewed thympoiesis and implementation of central tolerance B cell recovery was notably robust, with numbers comparable to healthy individuals for most patients by day 100. Subset analysis showed absence of memory B-cells but significantly increased proportion of immature transitional (T1 and T2) B cells (CD24+ CD38+, CD27-, IgM high IgD high) for 6 months after FCC HSCT. Cells within this subset are known to possess immune suppressive activity.The mechanism for prolonged mixed T-cell chimerism and associated low incidence of chronic GVHD following FCC conditioning for SAA HSCT may be multifactorial, but we here report novel findings: apart from a markedly low proportion of T cells, we show for the first time in SAA post HSCT the presence of T and B cell subsets with potential regulatory properties and establishment of central tolerance of naïve T-cells in younger patients. Our results also confirm the excellent outcomes of UD HSCT using alemtuzumab with reduced intensity conditioning in SAA, and we propose it is now time to re-think the up-front MUD transplant at all ages in eligible patients. DisclosuresNo relevant conflicts of interest to declare.

Use of High-Throughput Sequencing (HTS) of TCRß to Determine the Kinetics of Graft-Versus-Lymphoma (GVL) Effect and T-Cell Repertoire Profiles after Allogeneic Transplant

▪Introduction:While allogeneic transplant using non-myeloablative preparative regimen provides a viable alternative with low TRM, the kinetics of GVL effect and the T-cell reconstitution may differ from the myeloablative transplant. In general, the donor cell engraftment is slower using a non-myeloablative regimen and a delayed GVL effect is expected. In this study, we applied the TCRß HTS to determine (1) the kinetics of the GVL effect by quantifying the tumor cell burden prior to and after transplant and (2) the pace of the T-cell reconstitution after transplant.Method: This report included a cohort of 24 patients with advanced stage mycosis fungoides or Sézary Syndrome who underwent allogeneic transplant using a non-myeloablative regimen with total skin electron beam therapy (TSEBT, 24-36 Gy), total lymphoid irradiation (TLI, 8 Gy) and anti-thymocyte globulin (ATG). All patients received G-CSF-mobilized peripheral blood hematopoietic cells with a median donor CD34+ cell dose of 6.9 x 106/kg (range 2.0-12.4) and a median donor CD3+ cell dose of 278.1 x 106/kg (range 134.4-631.0). The unique malignant T-cell clonotype of each individual patient was identified from diagnostic blood/skin samples as the single dominant sequence by TCRß HTS. Blood samples were collected prior to and at different time points after transplant. DNA extracted from PBMC corresponding to approximately 200,000 genomes was used for HTS (Sci. Transl. Med.5:214ra171, 2013) (ImmunoSEQ, Adaptive Biotech). The tumor cell burden (including minimal residual disease, MRD) was expressed as percentage of the malignant clonotype found in the entire T-cell repertoire.Results: Prior to transplant, 21 patients (88%) had detectable disease by TCRß HTS in the blood (<1%: 10 patients, 1-5%: 3 patients, >5%: 8 patients), and 3 patients had no detectable disease in the blood. The percentage of malignant clone decreased in 19 of these 21 patients at day+30 post-transplant, and 2 patients showed stable minimal disease (0.03 and 0.30%, respectively). The reduction of tumor burden was most pronounced in patients with >5% involvement prior to transplant. In these 8 patients, the pre- and day+30 post-transplant disease burden decreased from 77.9 to 0.9%, 23.6 to 9.0%, 21.1 to 8.8%, 11.8 to 1.9%, 11.3 to 0.9%, 10.0 to 0.1%, 6.0 to 1.5% and 5.5 to 0.2%, respectively. This immediate post-transplant GVL effect was not associated with full donor T-cell engraftment (donor CD3+ >95%). The donor T-cell chimerism at day+30 was 94%, 79%, 1%, 90%, 93%, 93%, 23% and 91% for these 8 patients, respectively. Subsequently, 11 of the 24 patients achieved sustained molecular remission in the blood with a median time to achieve molecular remission of 60 days (range 30-540). Patients with full donor T-cell chimerism (n=16) had a higher chance of achieving molecular remission in the blood than those with mixed donor T-cell chimerism (69% vs 0%, p = 0.002). Of these 11 patients who achieved molecular remission in the blood, 8 also achieved molecular remission in the skin at the same time. Of the remaining 3 patients, 2 achieved molecular remission in the skin more than 4 months after achieving remission in the blood, while one patient has yet to achieve molecular remission in the skin.We then analyzed the “T-cell repertoire” at different time points post-transplant by assessing the number of unique T-cell clonotypes in each blood sample from 14 patients who had at least 1-year follow-up. While the size of the T-cell repertoire varied significantly between patients, we observed an overall upward trend within individual patients after transplant (Table). The size of T-cell repertoire did not correlate with the donor CD34+ or CD3+ cell dose in the allograft.Conclusion:By using an extremely sensitive and specific TCRß HTS, we have shown an immediate post-transplant GVL effect in which a full donor engraftment was not required, followed by a subsequent sustained GVL effect that may depend on full donor T-cell engraftment. Our results suggest a different kinetics of GVL effect in different compartments (blood vs skin). We also demonstrate continued expansion of T-cell repertoire profile after allogeneic transplant. Abstract 2473Table. The number of unique T-cell clonotypes at different time points post-transplantDay+30Day+60Day+90Day+180Day+270Day+360Median7,55010,4177,9428,40013,62820,057Range629 - 60,6441,916 - 63,6911,297 - 82,0591,572 - 66,5916,510 - 42,5651,914 - 57,358 DisclosuresNo relevant conflicts of interest to declare.

T-Cell Chimerism Is Valuable in Predicting Early Mortality in Steroid-Resistant Acute Graft-versus-Host Disease after Myeloablative Allogeneic Cell Transplantation

The main aim of this study was to evaluate the impact of early T-cell chimerism status on the incidence and clinical course of acute graft-versus-host disease (aGVHD) in allogeneic transplant recipients after myeloablative conditioning. Of 62 patients, 38 (61%) had complete T-cell donor chimerism (DC), and 24 (39%) had mixed T-cell chimerism (MC). The incidence of early death (before day 100) with steroid-resistant aGVHD was significantly higher in patients with complete DC compared to patients with MC (6/38 vs. 0/24, p = 0.04). The time from aGVHD diagnosis to nonrelapse death was markedly different in patients with complete DC versus MC [53 days (range 12-183) vs. 238 days (range 135-550), p = 0.005]. Conditioning with Etopophos/total body irradiation (TBI) resulted in significantly more patients with MC compared to conditioning with cyclophosphamide/TBI. In conclusion, early complete T-cell DC was associated with a high incidence of early death in patients receiving high-dose steroids for the treatment of aGVHD.

Decreased Risk of Acute Graft-versus-Host Disease Using Reduced Intensity Conditioning Compared to Myeloablative Conditioning is Independent of Donor-Recipient T-cell Chimerism

Background: It is not known if the reduced risk of graft-versus-host disease (GVHD) among patients receiving reduced intensity conditioning (RIC) as opposed to myeloablative conditioning (MAC) is due to differences in mixed donor-recipients chimerism, or to the intensity of the regimen. Methods: We compared patients with acute myeloid leukemia (AML) selected for RIC (n=47) to 46 patients selected for MAC before allogeneic hematopoietic stem cell transplantation (HSCT). Results: Time to neutrophils >0.5 x 109/L was median 15 days in the MAC group, which was faster than 17 days in the RIC group (p=0.001). MAC patients required more erythrocytes (p=0.001) and platelet transfusions (p=0.003). At four weeks, mixed donor-recipient T-cell chimerism was seen in 29% of the MAC patients and 46% of the RIC patients. Acute GVHD grades II-IV was 55% and 17% in the two groups, respectively (p<0.001). In multivariate analysis, acute GVHD was reduced using RIC (hazards ratio (HR) 0.23, p<0.001), for year of HSCT (HR 1.27, p=0.01), but not for mixed donor-recipient T-cell chimerism (HR 1.11, p=0.80). Transplant-related mortality (TRM) at three years was 15% versus 13%. Chronic GVHD and relapse were similar. Overall mortality was not affected by conditioning (HR 1.39, p=0.36). Discussion: To conclude, patients treated with RIC had an increased risk of acute GVHD as opposed to recipients of MAC, which was due to less intense conditioning and not due to mixed donor T-cell chimerism.

Early Mixed T-Cell Chimerism After Allogeneic Hematopoietic Stem Cells Transplantation Is Highly Predictive For Progression Free Survival

IntroductionChimerism analysis after allogeneic hematopoietic stem cells transplantation (HSCT) allows documentation and understanding of important clinical events such as engraftment, graft failure, and/or relapse. Few data are available on whether T-cell chimerism might be more informative than global chimerism. In this study, we focused on selective CD3+ T-cell chimerism and its interest to predict events after allogeneic HSCT. MethodsThis is a single center retrospective study. Only patients with a survival more than 3 months were included. Information concerning donors, recipients, conditioning regimen, graft harvesting and follow-up were collected in our center using prospective forms from Promise database. Evaluation of T-cell chimerism was performed on CD3+ blood cells after immunomagnetic sorting, by PCR-STR (short-tandem repeats) multiplex technique using 15 different markers. Analysis of the tandem repeat polymorphism was performed by sequencer and Genscan software (Applied Biosystem Inc.). Chimerism was evaluated in 2 ways: positive or negative, and % of recipient. An analysis with repeated measures was also performed to determine the utility of a positive measure of chimerism, independently of the date of sample after allograft, to predict a relapse. ResultsBetween January 2006 and December 2011, 148 patients (pts) were admitted in our unit for allogeneic HSCT (32% with myeloablative conditioning and 68% with reduced conditioning). Median age was 54.3 years, 59 male, 89 female. Diagnosis included AML/MDS (n=77), ALL (n=23), Aplastic anemia (n=4), lymphoma and CLL (n=23), myeloma (n= 8) and myeloproliferative disorders (n=13). The donor was matched sibling (n=51), matched unrelated (10/10) (n=74) or mismatched unrelated (9/10)(n=23). Graft source was PBSC (n=97), BM (n=33) or CB (n=18). At transplant, 108 pts (73%) were in complete remission, 19 (13%) presented partial response and 21 (14%) have progressive disease. At time of analysis, median follow-up was 1.75 years. Median overall Survival (OS) was estimated at 2 years. Forty-seven pts (32%) presented relapse. Seventy pts (47%) are still alive. Main causes of death were HSCT related (n=38), relapse or progression (n=33) and other (n=7). Acute GVHD grade II-IV incidence was 32% (13.5% grade III-IV) and 58% for chronic GVHD. At day +30, 56% of pts presented a mixed T-cell chimerism with 36 (24.3%) pts with a chimerism higher than 25% recipient. At day +90, 41% have remained positive. For NRM, aGVHD and age were the only prognostic parameters statistically identified. For PFS, only T-cell chimerism with a positive 25% cut-off at D+30 was identified as a prognostic factor (p=.0017, HR(IC 95%) 2.52(1.39; 4.58). PFS was not reached if T-cell chimerism was inferior to 25% at D+30 versus 1.35 years (figure 1). The results were concordant in a subgroup analysis performed according to the myeloid or lymphoid neoplasia status. Otherwise, cGVHD incidence was higher if T-cell chimerism was less than 25% at D+30 (70% vs. 18%). Finally, the analysis with repeated measures confirmed that positive T-cell chimerism is strongly correlated with relapse (p=.001; HR (CI95%) 90.18(30.11; 270.13)). ConclusionAfter allogeneic HSCT, mixed T-cell chimerism higher than 25% at day +30 is strongly correlated with poor PFS, independently of source of cells, donors, intensity of conditioning, diagnosis or disease status at transplant. Considering this high-risk population, early intervention should probably be considered. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cytomegalovirus Drives Mixed Chimerism Following T-Cell Depleted Allogeneic HSCT, Providing Protective Immunity In The Absence Of GvHD

A number of factors influence the incidence and degree of T-cell mixed chimerism following allogeneic stem cell transplantation (HSCT). The most well-recognized of these include donor source, GvHD and T-cell depletion of the graft. We hypothesized that in the ‘pro-tolerogenic’ environment following T-cell-depleted reduced intensity HSCT, viral infection could drive differential expansion of donor or recipient memory T cell (Tmem) populations, contributing to differences in the degree of mixed chimerism. Viral infection, more specifically cytomegalovirus (CMV) infection, has been shown to drive pauci-clonal T cell expansions in the early post-HSCT period, skewing T-cell repertoire reconstitution. Since CMV infection is associated with maintenance of relatively large memory T cell pools, and viral ‘reactivation’ can be associated with very large expansions of virus-specific T cells, we predicted that recipient T-cell chimerism would be greater following recipient seropositive/ donor seronegative (R+/D-) versus recipient seronegative/ donor seropositive (R-/D+) HSCT due to differential expansion of virus-specific Tmem. We evaluated the outcomes of 46 consecutive reduced intensity transplants incorporating in vivo alemtuzumab, in which these CMV serostatus combinations were present. Recipient chimerism was markedly greater in the R+/D- cohort (median 50% recipient, range 0-95% versus median 4%, range 0-95% respectively, P = 0.0044), and the correlation was greater when analysis was restricted to those with sibling donors (n=34, median 68% (2-95%) recipient versus 4% (0-95%) respectively, P = 0.0018), or to patients without grade II-IV acute or extensive chronic GvHD (n=33, median 63% (2-95%) recipient versus 2% (0-70%) respectively, P = 0.0019). At 6+ months post transplant, CMV-specific T cells accounted for 0.4-24% of the CD8+ T cell compartment. All such virus-specific T cells in R+/D- patients were recipient-derived, as demonstrated by chimerism analyses of CMV pp65-, pp50- or IE-1-specific HLA-streptamer-selected CD8+ T cells. Furthermore, chimerism was consistently more skewed towards recipient in the CD8+ as opposed to the CD4+ T cell compartment, in keeping with larger clonal burst sizes of CD8+ versus CD4+ virus-specific T cells. Since the majority of recipient-derived T cells were not CMV-specific, we hypothesize that events early post-transplant might have a role in ‘imprinting’ the level of recipient chimerism, which thereafter remains stable in the absence of GvHD. Interestingly, these recipient-derived CMV-specific T cells conferred apparent protection against multiple CMV reactivation episodes. The number of reactivation episodes and number of days on treatment within the R+/D- cohort were significantly higher for those with &lt;10% (n=9) versus those with &gt;10% (n=16) recipient chimerism (median 3 (range 1-10) versus 1 (1-3), P = 0.0044, and median 88 days (19-412) versus 21 days (17-63), P = 0.001, respectively). R+/D- transplants were not, therefore, generally associated with recurrent problematic CMV infection. Indeed, this scenario was largely confined to the group of patients developing GvHD, who suffered both from reduced absolute lymphocyte counts secondary to corticosteroids, and eradication of the recipient-derived T cell compartment including the CMV-specific Tmem pool. Paradoxically, this suggests that T-cell depletion may actually reduce the incidence of problematic CMV infection in this ‘high-risk’ group by facilitating persistence of recipient CMV-specific T cells. Twelve R+/D- patients received donor-lymphocytes at later time points for persistent recipient chimerism (median 78% recipient (5-95%)). All converted to&lt;5% recipient (median 0%). Serial analysis revealed rapid priming of donor-derived CMV-specific immunity in the absence of quantifiable CMV viremia (PCR became positive below the limit of quantification), with expansion of CMV-specific T cells accounting for up to 41% of the CD8+ compartment. In conclusion, these data indicate that in the absence of GvHD, CMV influences the pattern of chimerism following T cell depleted transplants, that recipient virus-specific T cells provide protection from recurrent CMV reactivation, and that conversion to donor chimerism following donor-lymphocytes is associated with clinically silent transition to donor-derived immunity. Disclosures: No relevant conflicts of interest to declare.

Acute GVHD is a strong predictor of full donor CD3+ T cell chimerism after reduced intensity conditioning allogeneic stem cell transplantation

The monitoring of chimerism is a standard procedure to assess engraftment and achievement of full donor lymphoid cells after reduced intensity conditioning (RIC) stem cell transplantation (Allo-SCT). However, there is no consensus on when and how often to monitor post-transplant chimerism. We retrospectively analyzed our experience regarding the impact of acute graft versus host disease (GVHD) for the prediction of allograft chimerism. One-hundred-and-fifteen patients transplanted between 2001 and 2010 were identified. This group included 57 females and 58 males with a median age of 50 years (range: 26-68). Patients evaluated in this study were adult patients with hematologic malignancies, who received transplants from an HLA-matched sibling donor or matched unrelated donor (MUD) at allele level so-called 10/10, and received the RIC regimen including fludarabine/busulfan and anti-thymoglobulin (ATG). Mixed T-cell chimerism was defined as between 5 and 94% recipient cells, and full chimerism was defined as the presence of more than 95% donor T-cell chimerism (TCC). Full donor TCC was achieved in 93 patients (81%) at a median of 77 days (range: 30-120) post-transplant. The cumulative incidence of Grade 2-4 GVHD in our population was 25% (95% CI 17-34). The analysis of the population of patients with acute GVHD grade ≥2 showed that at day 120 after Allo-SCT they all had a total full donor TCC. On the other hand, 78 (68%) patients without acute GVHD grade ≥2 presented with mixed chimerism (p = 0.002) on day 120 post-transplant. Interestingly, patients who received ATG 5 mg/kg obtained a higher probability of complete chimerism compared with those receiving 2.5 mg/kg (p = 0.03). In conclusion, our study demonstrates that acute GVHD was predictive of full donor TCC after RIC Allo-SCT. Therefore, our data may challenge the concept of the frequent or close monitoring of donor chimerism in some patients with ongoing acute GVHD. However, chimerism testing could represent an attractive modality for minimal residual disease detection or for impeding relapse warranting further prospective studies.