Chimerism Analysis Research Articles

Real-time qPCR for chimerism assessment in allogeneic hematopoietic stem cell transplants from unrelated adult and double umbilical cord blood

Allogeneic hematopoietic stem cell transplantation is the standard therapy for patients with various malignant hematologic disorders. A successful treatment results in complete engraftment of donor cells in the absence of the patient’s own hematopoietic system. Chimerism analysis, aimed at determining the coexistence of genetically different cell populations, is considered a useful method to measure treatment success. A new, qPCR based, commercially available chimerism assay was recently introduced. Here we report our results of comparing STR with qPCR-based chimerism analysis, and assessment of sensitivity and reproducibility of the qPCR chimerism assay. A specific emphasis is put on analyzing chimerism in recipients of double cord blood transplantation. We conclude that the qPCR chimerism assay for engraftment monitoring is a reliable and sensitive assay. Advantages and limitations of the assay in its current format are summarized.

Detection of relapse after hematopoietic stem cell transplantation in childhood by monitoring of WT1 expression and chimerism.

The measurement of minimal residual disease after hematopoietic stem cell transplantation is crucial for the prevention of hematological relapses. But many methods are limited to certain groups of patients. We present a comparison of two markers which are universally applicable: WT1 expression and chimerism status. We analyzed 1,848 peripheral blood and bone marrow samples of 148 children, adolescents and young adults with malignant hematological diseases. Median follow-up time was 739 days after transplantation. All patients suffering from hematological relapse showed high WT1 levels. Approximately half (51%) of the 37 relapses could have been detected early through an increase in WT1 expression. WT1 kinetics revealed to be more specific than single elevated WT1 levels (p < 0.05) and chimerism analysis (p < 0.05). Combined with chimerism analysis, 74% of relapses were detectable in advance. Elevated WT1 expression levels after transplantation reached the highest sensitivity (64.9%) as a single marker, although differences were not significant. When the dynamics of both markers as well as any elevated WT1 expression were taken into account, a sensitivity of 81.5% and a specificity of 83.7% were obtained. Hence, we conclude that WT1 is a useful marker for monitoring of minimal residual disease after transplantation, if specific targets are not available. WT1 expression and chimerism status should be mutually evaluated to decide about immunotherapeutic interventions aimed at preventing morphological relapse.

Application of Sensitive AlleleSEQR Chimerism PCR from Peripheral Blood in Allografted AML/MDS Patients Treated with Pre-emptive Posttransplant Immunomodulation

PCR technique based on short tandem repeats (STR, microsatellites) is currently most widely used to monitor chimerism after allogeneic hematopoietic stem cell transplantation. Analysis of the proportion of donor and recipient derived cells enables an early detection of graft rejection or secondary graft failure. In malignant diseases increasing recipient chimerism has also been used to predict relapse, particularly in patients who lack disease-specific PCR marker to detect minimal residual disease (MRD). In these patients serial chimerism analyses may guide the use of post-transplant pre-emptive immunomodulation with the goal of avoiding clinical relapse. However, the sensitivity of the recently standardized STR-PCR, ranging between 0.8 and 1.6%, may not be sufficient to allow detection of impending relapse early enough in rapidly proliferating diseases like acute myeloid leukemia (AML) [1]. Therefore, we have tested the Abbott AlleleSEQR chimerism analysis system in AML patients who received pre-emptive post-transplant immunomodulation. The patients were routinely investigated for chimerism with STR-PCR monthly from whole blood and from blood T-cell fraction during post-transplant period and from bone marrow CD34 positive precursor cell fraction at 2-3 month intervals. In high risk patients with AML or myelodysplastic syndrome (MDS), we have applied posttransplant immunomodulation with azacytidine, lenalidomide and/ or donor lymphocyte infusions (DLI) based on findings in MRD and chimerism analyses the target being MRD negativity and full donor chimerism. In this retrospective study the routinely collected samples were additionally analyzed by Abbott AlleleSEQR chimerism assay.

Laser Capture and Single Cell Genotyping from Frozen Tissue Sections.

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.

The Subfamily-Specific Assembly of Eag and Erg K+ Channels is Determined by Both the Amino and the Carboxyl Recognition Domains

The ether-a-go-go family of Kv channels encompasses three subfamilies: Eag, Erg, and Elk. Members of different ether-a-go-go subfamilies fail to form hetero-tetramers. Although a short stretch of amino acid sequences in the distal C-terminal section has been implicated in subfamily-specific subunit assembly, it remains unclear whether this region serves as the sole and/or principal subfamily recognition domain for Eag and Erg. Here we generated various chimeric constructs between rat Eag1 (rEag1) and human Erg1 (hErg1) subunits. Biochemical and electrophysiological characterizations of the subunit interaction properties of a series of different chimeric and truncation constructs over the C-terminus suggested that the putative C-terminal recognition domain is dispensable for subfamily-specific assembly. Further chimeric analyses over the N- terminus revealed that the N-terminal region may also contain a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the inter-subfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Interestingly, introduction of the homologous hErg1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 N-terminal chimeras. By contrast, the inactivation features were retained in the reverse hErg1 N-chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hErg1 eag domain fragment. Overall, our findings are consistent with the notion that both the N-terminal and the C-terminal recognition domains are required to sustain the subfamily-specific assembly of rEag1 and hErg1, and that the eag domain plays a critical role in regulating the inactivation gating of rEag1.

フローサイトメトリーを使用したキメリズム解析によるHLA不一致造血細胞移植後の病態解析

フローサイトメトリーを使用したキメリズム解析によるHLA不一致造血細胞移植後の病態解析

Loss of LKB1 leads to impaired epithelial integrity and cell extrusion in the early mouse embryo.

LKB1/PAR-4 is essential for the earliest polarization steps in Caenorhabditis elegans embryos and Drosophila oocytes. Although LKB1 (also known as STK11) is sufficient to initiate polarity in a single mammalian intestinal epithelial cell, its necessity in the formation and maintenance of mammalian epithelia remains unclear. To address this, we completely remove LKB1 from mouse embryos by generating maternal-zygotic Lkb1 mutants and find that it is dispensable for polarity and epithelia formation in the early embryo. Instead, loss of Lkb1 leads to the extrusion of cells from blastocyst epithelia that remain alive and can continue to divide. Chimeric analysis shows that Lkb1 is cell-autonomously required to prevent these extrusions. Furthermore, heterozygous loss of Cdh1 exacerbates the number of extrusions per blastocyst, suggesting that LKB1 has a role in regulating adherens junctions in order to prevent extrusion in epithelia.

Quantitative Monitoring By RT-PCR of Molecular Markers on Day +100 after Allogeneic Stem Cell Transplantation Predicts Outcome in Patients with Acute Myeloid Leukemia

Introduction:Monitoring of minimal residual disease (MRD) is commonly used after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML) patients. Flow cytometry of leukemia-associated aberrant immunophenotypes (LAIP) and chimerism analyses are frequently used tools to monitor MRD. These methods have lower sensitivities and lower relapse detection rates in contrast to molecular markers monitored by quantitative RT-PCR. However, their clinical implications are still limited and only little evidence of single molecular markers is available. We report on a retrospective study of 144 patients with molecular MRD markers transplanted at the Ludwig-Maximilans-University Hospital of Munich-Grosshadern, and analyzed the prognostic values of molecular MRD Monitoring after SCT, baseline risk factors, and follow-up (FU) markers (e.g. chimerism analyses and LAIP).Patients and Methods:Between January 2000 and August 2012, 495 AML patients underwent SCT at our institution. 164 patients had molecular MRD markers and 144 patients had at least one sample with quantitative results prior and/or after SCT, and were included into the analyses. At transplantation, patients had a median age of 48 years (range, 18 -73), and 49 patients were in complete remission, while 95 patients were in a more advanced status of their disease (>CR2). 106 patients received SCT from a HLA matched (10/10) and 38 patients from a HLA mismatched donor. Grafts were obtained either from related (n=60) or unrelated (n=84) donors. Most patients received reduced intensity conditioning (n=142). In 338 bone marrow samples MRD was monitored prior to SCT, on day +30 and on day +100. During the FU period after day +100, MRD was monitored at individual intervals in 429 peripheral blood samples. Quantitative RT-PCR was performed for NPM1 mutation (n=52), MLL-PTD (n=31), RUNX1-RUNX1T1 (n=12), CBFß-MYH11 (n=14), MLL rearrangements (n=20), MDS-EVI1/EVI1 (n=10), and DEK-CAN (n=5). Sensitivities of the different RT-PCRs assays ranged between 10-4 and 10-6.Results:After a median FU of 41 months (range, 4 – 115), 43 patients (30%) relapsed. The MRD levels monitored by RT-PCR prior to SCT and on day +30 after SCT showed no significant impact on relapse-free survival (RFS) and overall survival (OS). At day +100 after SCT, MRD positivity was strongly associated with worse RFS (HR 3.1, p=0.001) and OS (HR 3.2, p=0.004). This was also reflected in a cumulative two-year incidence of relapse of 61% for MRD positive patients versus 15% for MRD negative patients (p<0.0001). In addition, a change to MRD positivity during the FU period was strongly associated with a worse RFS (Mantel-Byar, p<0.0001). Furthermore, in univariate regression models we analyzed SCT baseline factors and FU markers. The remission status prior to SCT, the NPM1 mutation status, a HLA matched donor, the platelet count at day +100 (cut-off <50 G/l), and the European LeukemiaNet risk stratification (favorable versus others) were prognostic relevant risk factors. In the multivariate model only MRD positivity on day +100 (HR 3.7, p=0.013) and a low platelet count on day +100 (HR 5.0, p=0.005) were significantly associated with worse RFS. LAIP analyses (3-colors flow-cytometry, cut-off 0.1%) and a change in chimerism (STR-PCR and XY-FISH, cut-off 80% and 90%) of sorted (CD3+) and unsorted cells at the respective time points had no influence on RFS and OS.Conclusions:Molecular MRD monitoring after SCT might be a useful tool to identify AML patients at high risk for relapse, in contrast to other FU markers, e.g. chimerism analyses and LAIP. In particular, MRD positivity on day +100 after SCT and the switch to MRD positivity during the FU period were significantly associated with worse RFS. As perspective, the individual molecular MRD course might be used as prognostic factor for the guidance of treatment post SCT. Patients with MRD positivity on day +100 after SCT or with a switch to MRD positivity in the FU period may be considered for donor lymphocyte infusions (DLI) or chemotherapeutic interventions, such as hypomethylating agents. DisclosuresSubklewe:AMGEN Inc.: Research Funding.

Long-Term Outcome of Preemptive Immunotherapy Based on Post-Transplant Chimerism and MRD Monitoring after Allogeneic Stem Cell Transplantation in Children with Acute Lymphoblastic Leukemia: A Single Center Experience

Introduction:Mixed chimerism (MC) and minimal residual disease (MRD) strongly predict risk for relapse in children with acute lymphoblastic leukemia (ALL) following allogeneic stem cell transplantation (allo-SCT). Preemptive immunotherapy (IT), e.g. withdrawal of immunosuppression (WD-IS) or donor lymphocyte infusion (DLI) guided by chimerism and MRD monitoring can prevent impending relapse in allo-SCT recipients. In this study we retrospectively analyzed chimerism and MRD monitoring and the effect of preemptive IT in all pts undergoing allo-SCT for ALL in our institution.Patients:Between January 2005 and July 2014, a total of 89 children and adolescents (median age 11.5; range 2.2-26.0 years) with ALL (pB-ALL, n=63; T-ALL, n=20; biphenotypic/bilinear ALL, n=6) were transplanted in our center after remission induction treatment. 47 pts were in first, 26 pts were in second, 15 pts were in third, and 1 pt was in fourth complete remission (CR) at time of transplant. 18 pts received grafts from matched sibling donors (MSD), 61 pts from matched unrelated donors (MUD), and 10 pts were grafted from a haploidentical parent. Pts who received their graft from a matched donor (n=79) all underwent a fully myeloablative conditioning regimen consisting of TBI (12 Gy) and etoposide. Pts who were grafted from a haploidentical donor were prepared with fludarabine, thiotepa and melphalane.Methods:Serial and semi quantitative analyses of post-transplant hematopoietic chimerism in peripheral blood were performed weekly until day 200 and monthly thereafter. Bone marrow analyses were done at days +30, +60, +90, +180 and +365 post-transplant. Thereby, MRD was assessed in 56 pts, whereas no diagnostic material was available in 33 pts. Standardized quantification of MRD was performed according to the guidelines of the Euro-MRD-Group. All patients should receive preemptive IT if they develop MC after day +28.Results:64/89 pts (72%) showed complete chimerism (CC) in all follow-up analyses, and 25/89 pts (28%) developed MC although all pts received a fully myeloablative conditioning regimen. From 56 pts in whom MRD could be assessed, 40 pts remained MRD negative and 16 developed MRD positivity after transplantation. IT (WD-IS, n=12; DLI, n=14) was initiated based on chimerism analysis in 22/25 pts with MC, and was guided by MRD detection in 4 pts.For the total cohort of pts, probability of event free (pEFS) and overall survival (pOS) were 0.67 and 0.77, respectively. Furthermore, cumulative incidence of treatment related mortality (CI-TRM) and relapse (CI-relapse) were 0.11 and 0.24 for all pts.Chimerism:pEFS was 0.74 in CC-pts and 0.51 in MC-pts (p<0.032). 22/25 MC-pts received pre-emptive IT resulting in a pEFS of 0.58. Due to rapid progression of their disease IT was not initiated in 3/25 MC-pts. All MC-pts without IT relapsed, and in part received further treatment. While CI-TRM remained low (0.10 for CC resp. 0.12 for MC, p>0.68) in both groups, CI-relapse was 0.17 in CC- and 0.41 in MC-pts (p<0.021).MRD:Besides MC, MRD level post-transplant emerged as poor prognostic factor. Pts were grouped according to their highest MRD value post-transplant in MRD negative, low positive (<10E-4), and high positive (>10E-4) pts. pEFS and pOS were 0.82 and 0.95 in MRD negative (n=40), 0.56 and 0.88 in low level MRD (n=8), and 0.25 and 0.25 in high level MRD (n=8) positive pts (p<0.001). CI-relapse was 0.14 in MRD negative, 0.36 in MRD low, and 0.75 in MRD high positive pts (p<0.001), while CI-TRM between these groups remained low (0.05 for MRD negative, 0.13 for MRD low and 0.00 for MRD high, p>0.55).Other risk factors:Furthermore, remission status at the time of transplant influenced relapse rate. CI-relapse was 0.16 in CR1, 0.26 in CR2, and 0.45 in CR>2 pts (p<0.025). Differences in pEFS, pOS and CI-TRM among CR1, CR2, and CR>2 pts were not statistically significant.Multivariate analysis:Multivariate analysis for pEFS also indicated that MC and high level MRD were independent poor prognostic factors (MC, p<0.049, RR 3.16 and high level MRD, p<0.022, RR 3.99), while remission status before transplantation, ALL lineage, donor type, graft source, T cell depletion or sex showed no significant influence.Conclusion:Our results show that analysis of chimerism and MRD allow the prediction of impending relapse in virtually all children and adolescents transplanted for ALL and that preemptive IT can improve outcome in these high-risk pts. DisclosuresNo relevant conflicts of interest to declare.

Lineage-Specific Chimerism Monitoring Using Flow Cytometry with Anti HLA-Antibodies in Haploidentical Hematopoietic Stem Cell Transplantation

Introduction: Haploidentical hematopoietic stem-cell transplantation (HHSCT) is an alternative transplant strategy for patients who lack a suitable HLA-matched donor. Determining the degree of engraftment can be critical to establishing the success of a HHSCT. Short tandem repeats-PCR (STR-PCR) based chimerism analysis is widely used, but most has been performed by testing total leukocytes. Analysis of chimerism in selected lineage-specific populations could provide more important information. However, STR-PCR of isolated cells is labor intensive and time consuming. In HHSCT settings, HLA antigens were used as markers to establish the presence of chimerism. Flow cytometry can be used to evaluate chimeric status in HHSCT based on disparity of HLA antigens between donor and recipient. In this study, we aimed to observe the dynamics of changes in cell subpopulations and engraftment after HHSCT with flow cytometric lineage-specific chimerism analysis (FCM-LSCA).Methods: Peripheral blood samples of total sixty-nine pediatric patients who underwent HHSCT in Asan Medical Center were collected between October 2011 and June 2014. Diagnoses were aplastic anemia (n=27), acute myeloid leukemia (n=21), acute lymphoblastic leukemia (n=10), non-Hodgkin’s lymphoma (n=7), non-malignant hematologic disorders (n=4). Four patients received CD34-selected graft, 26 patients received CD3-depleted graft, and 39 patients received TCRαß-depleted graft. Patients who experienced graft loss or disease relapse were six. FCM-LSCA performed using antibodies for HLA antigens and cluster of differentiation (CD) antigens. We selected a panel of specific HLA antibodies to target HLA serotypes based on the allele distribution of HLA in Korean population. FCM-LSCA of T cell and NK cell was done according to regularly scheduled protocol from the start of stem cell infusion (PID0).Results: Fifty-two patients (75% of total patients) showed suitable anti HLA-antibodies for FCM-LSCA. The proportion of donor NK cells increased sharply and reached 100% of lymphocytes at PID 2-3 in the second or third HHSCT. In comparison, donor NK cells reached 100% of lymphocytes approximately 3 days after the first successful HHSCT. The proportion of donor NK cells reached 100% but decreased and reached 0% at PID 14-21 in failure of engraftment. CD56 dim/CD57 positive NK cells among CD56 positive cells reached 50% at PID 3 and remained over 50% until PID 22 and slowly decreased to 40% at PID 35. CD56 dim/CD57 negative cells had highest expression at PID 18 and decreased slowly. CD56 bright/CD16 negative cells reached peak proportion at PID 28, comprising 20% of CD56 positive cells. Among the gated lymphocytes, the median percentage of donor T cells at PID 10 of patients who experienced graft failure was significantly lower than that of patients who achieved sustained engraftment [5.5% versus 88.7%, P=0.000]. During scheduled monitoring, we detected mixed chimerism of T cell with increased recipient cells in two acute leukemia patients. Emergent bone marrow examination performed and diagnosed relapse of leukemia. Two of aplastic anemia patients showed complete donor chimerism on STR-PCR (more than 95% donor cells), but showed persistent mixed chimerism on T-cell LSCA (22.9% and 25.8% of recipient cells, respectively).Conclusions: Serial monitoring of FCM-LSCA will give a better understanding of regenerating hematopoiesis after transplantation as well as engraftment or rejection kinetics. In practical consideration, preliminary evaluation of anti-HLA antibody reactivity in donor and recipients will be essential. Especially, our study strongly suggests that monitoring FCM-LSCA is useful to enable early diagnosis and effective treatment of impending graft rejection or recurrence of original disease in HHSCT. DisclosuresNo relevant conflicts of interest to declare.

A New Strategy to Overcome the Cell Dose Barrier to Umbilical Cord Blood Transplants: A Proof of Early Hematopoietic Reconstitution By Combined Multiple Units of Allogeneic Stem/Progenitor Cells

Introduction: Umbilical cord blood (UCB) serves as a suitable donor source in hematopoietic stem cell transplantation (HSCT). However, UCB has the major drawback that is delayed engraftment due to its low graft cell numbers, which often limits its use in HSCT. To overcome this cell dose barrier, double units UCB transplantation (UCBT) has been attempted, but the time to engraftment is still relatively delayed. Based on the report by Japan Red Cross Society, the majority of UCB units remain unused clinically because of their insufficient graft cell doses. Overall, these facts prompted us to seek for a new strategy to improve UCBT by using multiple (more than three) units. We here provide a proof of feasibility of such an approach using mouse transplantation models.Methods: To mimic a clinical setting of UCBT, we first established an "insufficient cell dose" model by using mouse BM KSL (c-Kit+, Sca-1+, lineage-marker-negative) cells. In this model, C57BL/6 (B6-Ly5.2, H2b) mice were the recipients, and congenic B6-Ly5.1 mice (H2b) were the primary donors of cell grafts. We used KSL cells as a unit of grafts, as they contained hematopoietic stem and progenitor cells (HSPC) that were able to restore BM reconstitution. KSL cells were used also to avoid inclusion of mature immune cells, which could compromise the outcomes in HSCT through unpredictable immune reactions. As the donors of combined multiple units, four allogeneic mouse strains were used: BDF1 (DBA2 x B6 F1, H2b/d), B6D1F1 (DBA1 x B6 F1, H2b/q), B6C3F1 (C3H x B6 F1, H2b/k), and CBF1 (Balb/c x B6 F1, H2b/d). The use of F1 strains was to prevent GvHD, which would significantly affect donor cell engraftment kinetics. The recipient mice were lethally irradiated, and thus could not survive with only the "insufficient cell dose" of B6-Ly5.1 graft. The outcomes in HSCT were tested by the addition of mixed allogeneic KSL cells (multi-allo HSPCs) in comparison with the addition of B6-Ly5.1 KSL cells (congenic HSPCs) with the equivalent doses. The effects of multi-allo HSPC transplants were evaluated by recipients’ survival rate and complete blood counts over time. Detailed donor cell contribution in peripheral blood and BM was also determined by flow cytometry analysis. The transgenic mouse line expressing a fluorescent marker Kusabira Orange was utilized where needed to test donor cell contribution in erythrocytes and platelets.Results: Titration studies revealed 500 B6-Ly5.1 KSL cells as optimal for the "insufficient cell dose" model because the recipients were all lethal, whereas ~100% mice survived upon receipt of 2,500 B6-Ly5.1 KSL cells. Interestingly, addition of multi-allo HSPCs rescued otherwise lethal recipients as effectively as congenic-HSPCs with the equivalent acceleration of hematopoietic recovery. Chimerism analysis, however, revealed that this "KSL cells alone" transplantation led to long-term existence of multi-donor hematopoiesis, which was not ideal for a clinical setting. We then replaced B6-Ly5.1 KSL grafts with the whole BM (WBM) grafts. Titration experiments determined 50,000 WBM cells as a single unit, mimicking an "insufficient dose" of unmanipulated UCB unit. Addition of multi-allo HSPCs in this model also showed complete protection of recipients from lethality and enhanced early hematopoietic recovery (figures). Remarkably, dominant B6-Ly5.1 chimerism was established and maintained in this modified model. Experiments using subfractionation of the B6-Ly5.1 grafts demonstrated that small numbers of T cells were responsible to single-donor chimerism formation possibly through graft versus graft reactions. Finally, to maximize transient early hematopoietic reconstitution by multi-allo HSPCs (we call this "bridging effect"), we tested whether cultured hematopoietic stem cells (HSC, defined as CD34negatvie/low KSL cells) worked better than uncultured KSL cells. Using our defined protocol compatible with stem cell amplification, we demonstrated that a mixture of cultured allo-HSCs exhibited the bridging effect even superior to that of an uncultured KSL cell mixture.Conclusions: We here provide a proof that combined multiple units of allogeneic HSC/HSPC are capable of exhibiting "bridging effect" when manipulated appropriately and added to an otherwise "insufficient dose" setting of transplantation. Development of a new strategy of multiple units transplantation may open a door for wider application of UCBT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Graft Versus HIV-1 Reservoir Effect after Allogeneic Stem Cell Transplantation

Background: The use of a stem cell source homozygous for the CCR5 delta32 mutation is of particular interest in HIV+ patients in need of an allogeneic hematopoietic stem cell transplantation (SCT) given the high natural resistance against HIV-1 acquisition. In the absence of such cell source, the impact of allogeneic SCT on human immunodeficiency virus type 1 (HIV-1) reservoirs in patients receiving combination antiretroviral therapy (cART) is unknown.Methods: Size of HIV-1 reservoirs was investigated in 3 patients with hematological malignancies receiving cART, who underwent allogeneic SCT (Table 1). Patient 1 received a myeloablative single cord blood (wild type (WT) CCR5), supported with third party HLA-mismatched CD34+ cells (haplo-cord SCT). Patients 2 and 3 underwent reduced intensity conditioning SCT of an HLA-matched sibling (heterozygous CCR5 delta32 donor and WT CCR5 donor, respectively). In-depth analysis of the HIV-1 reservoir was performed in the post-transplant period using droplet digital PCR and hemi-nested qPCR (proviral DNA copies/million PBMC). Chimerism analysis was performed by short tandem repeat PCR (STR-PCR).Results: Proviral DNA showed a decreasing dynamics after allogeneic SCT and became undetectable in all 3 patients at 9, 18 and 1 months, respectively (Table 1). In patient 1, negativity of proviral HIV-1 DNA coincided with achievement of complete chimerism while being free of immunosuppression. Patient 2 showed undetectable proviral DNA together with the development of skin chronic GvHD. Finally, undetectable proviral DNA and complete chimerism was achieved in patient 3 early after transplantation. All patients remained in complete hematological remission.Conclusion: All our patients showed a small HIV-1 reservoir after allogeneic SCT. The coincidence of achievement of negative proviral HIV-1 DNA and complete chimerism and/or development of GvHD suggests a role of Ògraft versus HIV-1 reservoir effectÓ in the control of HIV infection. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stable Blood Group Chimerism in Recipients of ABO Incompatible Allogeneic Stem Cell Transplantation

Background: The ability to express the ABH antigens in oral mucosa and saliva is present in about 80% of the general population (hence called secretors) and is regulated by the Sec gene on chromosome 19q13.3, which encodes for the enzyme fut2 (α(1,2)Fucosyltransferase), resulting in the expression of blood types in body fluids, including saliva. The gene encoding for the ABH antigens that determines ABO blood type, is located on chromosome 9q34, and transcribes to the enzyme fut1. While fut1 is mainly expressed in erythroid tissue, fut2is expressed in secretory cells. The aim of the present study was to evaluate the ABO type in saliva and red blood cells of patients undergoing allogeneic SCT from ABO mismatched donorsMethods: Secretion status and ABO type in saliva and blood were analyzed in patients with different hematological malignancies undergoing alloSCT from ABO incompatible donors and from healthy donors serving as controls. All study participants signed informed consent. For the determination of ABO type in saliva, following mouth rinse, 5 cc of saliva were collected from each participant into fresh tube and immediately frozen at -800C. Saliva ABH antigens were extracted and enzymes were inactivated. Secretor status and ABO type in patients’ saliva were determined by inhibition test. Agglutination of diluted A, B or O typed donor red cells was tested macroscopically in the presence or absent of the extracted ABH antigens pre-incubated with anti-A, anti-B or anti-H, respectively. ABO blood type was routinely determined in patients at least every 2 weeks and time to ABO type conversion was recorded as along with all transplant-related clinical dataResults: The study cohort included 30 patients (16 males and 14 females; median age 54.2, 18.8-68.5), who underwent alloSCT between Dec 2009 and Feb 2014 from an ABO incompatible donor and were available for routine follow-up. Median follow-up time from transplant to last ABO determination in saliva was 613 days (153-2789).Donors were matched related in 11, matched unrelated in 16 and mismatched unrelated in 3 cases. All grafts were from mobilized peripheral blood. Transplant from major, minor and bidirectional ABO incompatible donors was present in 9, 16 and 5 recipients, respectively. All patients engrafted. Chimerism analysis at day 30 and 100 post transplant by PCR for STR was 100% in 24/25 and 23/25 of tested patients, respectively. Median days to ABO type conversion were 64 (21-290). Of 30 patients, 26 were found to be secretors (87%).In the secretor group, 29/30(96.6%) retained original blood group in the saliva, while one patient originally typed as AB and transplanted from an A type donor, did not retain his original AB blood type in the saliva. It is not clear whether the lack of B-antigen is attributed to the acute mucosal GvHD or to a true conversion of the ABH antigen expression in the salivaConclusion: To the best of our knowledge, this is the first report of stable chimerism of the ABO blood groups post ABO-incompatible allogeneic transplantation, such that the majority of recipients (96.6%) retained the recipient ABO group in the saliva, while expressing the donor ABO group in the blood. The significance of these findings and correlation with long-term outcome need to be further studied in larger patient cohort DisclosuresNo relevant conflicts of interest to declare.

Allogeneic Peripheral Blood Stem Cell Transplantation Using Reduced-Intensity Conditioning Regimen with Fludarabine and Busulfan from HLA-Matched Related Donor for Elderly Adult T-Cell Leukemia/Lymphoma: Results of Multicenter Phase II Study (ATL-NST-3)

Background: Adult T-cell leukemia-lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type 1 (HTLV-1) infection and commonly affects individuals at an average age of 67 years. Since the prognosis of aggressive ATL patients (pts) treated with conventional chemotherapy has been dismal, we had so far conducted two clinical trials to evaluate the feasibility of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an HLA-identical sibling donor using reduced-intensity conditioning regimen (RIC) consisting of fludarabine and busulfan. We reported that allo-PBSCT using RIC provided a long-term survival in about one third of ATL pts and that it exerted not only graft-versus-ATL effects but also graft-versus-HTLV-1 effects (Okamura J, et al. Blood 2005; Tanosaki R, et al. Biol Blood Marrow Transplant 2008; Choi IL, Bone Marrow Transplant 2011). To confirm these promising results in a larger number of ATL pts, we conducted a phase II study (UMIN C000000409).Patients and Methods: Between September 2006 and July 2011, 20 pts were registered in this study. Because ATL is a rare disease affecting elderly people, their siblings were also old and were not appropriate for donors, and the enrollment of pts needed much more time than we had expected. Therefore, though we had originally planned to enroll 35 pts, we had to close this study incompletely. The conditioning regimen consisted of fludarabine 30 mg/m2/day intravenously days -8 to -3, and busulfan 3.2 mg/kg/day intravenously or 4 mg/kg/day orally days -6 and -5. GVHD prophylaxis was CsA alone from day -1 and PBSCs (CD34+ cells >2x106/kg) were infused on day 0. Primary endpoint was 2-year overall survival (OS). Engraftment, adverse events such as graft-versus-host disease (GVHD) and infectious events, and progression free survival (PFS) were also evaluated as well as donor chimerism by STR-PCR analysis, HTLV-1 proviral load, and Tax-specific cytotoxic T lymphocytes (CTL) by tetramers. The eligibility criteria at registration included acute- or lymphoma-type ATL, pts with an HLA-matched related donor, age between 50 and 70, disease status of CR, PR or NC with stable disease, no uncontrollable active infections, no major organ dysfunction, and no active CNS involvement.Results: Median age was 54 years (range 50-68), genders were 10 males and 10 females, ATL types were 13 acute and 7 lymphoma, and disease status at transplantation was 9 CR, 8 PR, and 3 NC. 13 donors were HTLV-1 positive. Engraftment as confirmed by donor chimerism analysis was achieved in all pts. OS and PFS were 95% (95%CI, 86-100%) and 60% (95%CI, 39-82%) at day 100, 74% (95%CI, 55-94%) and 45% (95%CI, 23-67%) at 1 year, and 53% (95%CI, 31-76%) and 40% (95%CI, 19-62%) at 2 years. Acute GVHD occurred in 13 pts (grade I in 2, II in 5, III in 6 pts), and chronic GVHD developed in 12 pts (63%) out of 19 evaluable pts. Eleven pts died at the time of analysis; ATL 6, GVHD 3, IP 1, PCP 1. There was no significant relationship between OS and grades of acute GVHD. While HTLV-1 proviral load decreased as early as 1 month after transplantation and became undetectable level in 14 pts (70%) out of 20, the increase of A2- or A24-restricted Tax-specific CTLs in HLA-A2 or A24-positive pts, respectively, were observed in some pts after 3 months or later, and there was no clear relationship between them. With a median follow-up of 36 months (range 24-86), 9 pts (5 CR and 4 PR at transplantation) were alive without disease.Conclusions: It was confirmed that allo-PBSCT using RIC consisting of fludarabine and busulfan was relatively safe and effective, and provided a long-term survival at least in chemo-sensitive aggressive ATL pts. DisclosuresNakamae:Novartis: Honoraria, Research Funding, Speakers Bureau, Travel/accomidations/meeting expenses Other.

Engraftment Effects of Intra Bone Marrow Compared to Intravenous Transplantation of Allogeneic Hematopoietic Stem Cells Following a Reduced Intensity Conditioning in a DLA-Identical Canine Littermate Model

Introduction: Successful engraftment following hematopoietic stem cell transplantation (HSCT) depends on factors like immunosuppression, graft composition and number of infused HSC. Whereas the immunosuppression as well as the type and composition of the graft are influenceable low numbers of available HSCs i.e. “weak grafts” remain a clinical challenge. Weak grafts are accompanied by increased graft failure rates and longer cytopenias associated with increased morbidity. Intra bone marrow (IBM) infusion of HSC might be an approach to overcome these problems. Studies in rodents demonstrated faster engraftment with an IBM HSCT approach compared to intravenous (IV) HSCT following myeloablative conditioning. Studies of IBM HSCT following non-myeloablative or reduced intensity conditioning (RIC) are missing.Aims: Exploring the feasibility and efficiency of IBM allogeneic HSCT in comparison to IV HSCT in dog leukocyte antigen (DLA) identical canine littermates using a RIC regimen.Methods: DLA-identical siblings were used as donor/recipient pairs for HSCT. Recipient dogs were conditioned with 4.5 Gy total body irradiation before HSCT (d0) and received 15 mg/kg Cyclosporin A BID as pre- and postgrafting immunosuppression (d-1 to d+35). BM grafts were harvested at d0. In the control group (CON, n=7) unmodified BM was transplanted IV. In the IBM group (n=7) BM harvests were centrifuged and buffy coat of the BM was then transfused simultaneously into the recipient humeri and femura (50 ml, 10 min). 10 dogs are currently evaluable. Chimerism of the peripheral blood mononuclear cells (PBMC) and granulocytes (G) were tested weekly until week 8 and afterwards in larger intervals. Blood cell counts and clinical toxicities such as weight loss were monitored.Results: Infusion of BM directly into the bone was feasible. All animals engrafted. Median number of infused total nucleated cells was 4.0*108/kg (range 2.3-6.0*108/kg, IBM) and 3.3*108/kg (range 1.9-5.0*108/kg, CON, IBM vs CON: p=0.4). Median CD34+ numbers infused were 3.1*106/kg (range:1.2-10.0*106/kg, IBM) and 3.9*106/kg (range: 1.0-7.2*106/kg, CON; IBM vs CON: p= 0.8).Hematopoietic recovery in the IBM and CON groups were similar. Leukocytes recovery (>1.0*109/l) occurred at median d+11 (range: d+10 - d+16, IBM) and d+10 (range: d+9-d+12, CON; IBM vs CON: p=0.3). Median leukocytes nadirs amounted to 0.23*109/l (IBM) and 0.28*109/l (CON; IBM vs CON: p=0.3) and median duration of leukopenia (<1.0*109/l) were 6 days (range: 5.0–11.0, IBM) and 4 days (range: 3.0–6.0, CON; IBM vs CON: p=0.1). Median platelet nadir after IBMT was 10.0*109/l (range: 0.0 - 25.0*109/l) and 6.0*109/l (range: 3.0-15.0*109/l, CON; IBM vs CON: p=0.8). Period of thrombocytopenia (≤50.0*109/l) lasted for 12 days in both groups (p=0.7).Chimerism analyses showed an early and fast increase in donor chimerism in both groups. The PBMC donor chimerism at d+14, d+28 and d+56 were 46% (range: 30-53%), 57% (range: 40-73%), 64% (range: 60-83%) for IBM. Results in CON were 37% (range: 17-93%), 60% (range: 49-100%), 57% (range: 40-100%) (IBM vs CON, p=n.s. (all time points)). The G chimerism values at that specific points were 95% (range: 53-100%), 100% (range: 53-100%), 96% (range: 88-100%) for IBM and 100% (range: 93-100%), 99% (range: 92-100%), 98% (range: 93-100%) for CON (IBM vs CON, p=n.s. (all time points)).Primary goal of the study was the feasibility of the IBM approach. Ethics regulations did not allow to use weak grafts (≤2.0*106/kg) intentionally. However, 4 animals received weak grafts (CON n=2, 1.0 and 2.0*106/kg; IBM n=2, 1.2 and 1.3 *106/kg). Of interest, comparing data of these dogs showed that durations of leukopenia were similar (median 10 days, both groups), but duration of thrombocytopenia were different (median 8 days, IBM vs 22 days, CON). Additionally, long term donor chimerism was higher in the IBM (median 80% PBMC, 100% G) vs CON (median 61% PBMC, 42% G).Conclusion:First, IBM HSCT is a feasible and effective method to deliver HSC directly into the bone marrow following RIC in a canine HSCT model. Second, our preliminary data suggest that IBM HSCT reveals advantageous engraftment differences in regards to platelet recovery and donor chimerism kinetics compared to the IV HSCT when grafts with low HSC numbers were infused. Follow up data of this study and future studies will have to clarify these observations further. DisclosuresNo relevant conflicts of interest to declare.

Haplo-Cord UCB SCT with Low Cell Dose, Well Matched UCB Units. a Prospective Multicenter Study

The outcome of umbilical cord blood transplantation is governed by UCB cell dose and matching. But stringent cell dose requirements limit access to well matched units. In haplo –cord transplant, early engraftment is determined by the adult graft. Elsewhere we have shown that excessive doses of haplo-identical cells resulted in failure of the UCB graft. Here we investigated the effect of a lower UCB cell dose threshold on ability to identify well matching units. We also prospectively evaluated the effects of UCB cell dose and matching (combined with a fixed dose haplo-graft ) on cord engraftment, platelet recovery, neutrophil recovery and acute GVHD, PFS and OS.We enrolled three consecutive cohorts with increasingly lower UCB cell dose thresholds (1) UCB cell dose >2x 10^ NC/kg (2)≥1x 10^ NC/kg and (3)≥.5x 10^ NC/kg. Additional selection criteria included (1) best HLA match (initially based on 6 HLA, as of mid 2013 based on 8 HR antigens (2)UCB viability, (3) avoidance of DSA antibodies and (4) NIMA compatibility. We continued enrollment on each cohort until at least 10 patients were evaluable for day 100 chimerism. If more than 7 of ten evaluable patients in a cohort achieved >40% UCB chimerism by d 100, the next cohort was started. This rate of success was achieved in all three cohorts, so we have continued accrual in the lowest cell dose. Sixty patients have been accrued at two institutions over 19 months. 43/60 pts had AML or MDS. The majority had ASBMT advanced and intermediate disease. Nearly half of the pts were of minority descent. Conditioning in all pts consisted of fludarabine-melphalan (three pts also received TBI 400) and GVHD prophylaxis of tacrolimus and mycophenolate. Haplo identical CD34 selected cells were co-administered at a dose of 3-5 x10^6 CD34 cells/kg. Pt characteristics and preliminary outcomes are summarized in the table below.TableCohort123Minimum TNC x10^5/kg210.5n152025Age486264ASBMT Low/Int/Adv4/5/67/5/78/6/11Weight (kg)73 (56-96)72 (50-136)85(46-122)Caucasian/Other7/813/712/13Median UCB TNC collected (x10^7/kg) and range2.7 (2.1-8.3)2.1 (1.4-2.9)1.7 (0.9-2.7)UCB TNC/kg>3x10^7/kg7 (46%) (0 UCB failure)1 (5%) (0 UCB failure)2 (8%) (0 UCB failure)2-3 x10^7/kg8 (54%) (0 UCB failure)9 (45%) (2 UCB failure)(32%) (0 UCB failure)1.5-2 x10^7/kg05 (25%) (0 UCB failure)8 4 (16%) (0 UCB failure)1-1.5 x10^7/kg05 (25%) (0 UCB failure)10 (40%) (4 UCB failure)<1 x10^7/kg001 (4%) (1 UCB failure)High Resolution HLA match8/801 (5%)4 (16%)7/82 (6%)7 (35%)7 (28%)6/803 (15%)8 (32%)5/86 (40%)9 (45%)5 (20%)<5/88 (54%)01 (4%)Median Time to ANC>500 (range)14 (10-60)12 (9-18)12 (9-51)Median Time to Plt >20 (range)35 (15-88)20 (13-NR)21 (10-NR)% with >40% UCB chimerism @d1001008376* Analysis of UCB graft characteristics includes all patients. Engraftment and GVHD analysis excludes 10 patients with early fatal complications or relapse. Chimerism analysis excludes two additional patients with missing information. NR= not reachedThe average HLA matching was significantly better (P<.001) in groups 2 and 3 (66% with >6/8 match), than in group 1 (6% with >6/8 match). UCB cell doses were significantly lower in groups 2 and 3 (55% with UCB cell dose <2x10^7/kg vs none in group 1). There were no significant difference between the groups in hematologic recovery, OS, PFS or UCB engraftment.Seven of 50 evaluable pts had failure of the UCB graft (<40% UCB chimerism on d 100). 4 of them had 100% haplo chimerism, 2 had autologous recovery and 1 had partial <40% UCB chimerism. The incidence of UCB failure increased with decreasing UCB cell dose (P<0.001 U test), with five of seven graft failures occurring in patients receiving UCB doses <1.2 x10^7/kg.This novel cord blood selection algorithm allows identification of well-matched UCB cells (at least 6/8 HR) in two thirds of patients. In approximately one third of cases the best matching UCB unit has a cell dose <1.5 x10^7/kg. Very low UCB cell doses appear to be associated with somewhat increased risk of UCB graft failure-delay. Long-term follow up will establish the impact of this selection algorithm on OS, PFS, chronic GVHD. Disclosuresvan Besien:Miltenyi Biotec: Research Funding. Stock:Sigma-Tau: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Mark:Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millennium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding, Speakers Bureau.

High Rates of Early Donor Chimerism in Intermediate and Poor Risk Patients Undergoing Stem Cell Transplantation Using Reduced Toxicity Ablative Conditioning Regimen Incorporating Busulfan Pharmacokinetics

▪Reduced toxicity ablative conditioning regimens are increasingly used for allogeneic stem cell transplantation (allo-SCT). The impact of early donor cell chimerism on outcomes of T cell–replete reduced-intensity conditioning SCT in myeloid disorders is ill defined. To explore the impact of measuring busulfan pharmacokinetics in conditioning regimens on early donor chimerism, we undertook a retrospective analysis of patients with myeloid disorders who received four days of fludarabine and busulfan with or without measuring busulfan pharmacokinetics at our center in the last 10 years.Methods: Chimerism assay was performed using a quantitative fluorescence-based short tandem repeat– polymerase chain reaction (STR-PCR) with capillary electrophoresis for PCR product resolution.Results: Thirty patients were identified and included in the analysis. All patients were conditioned with fludarabine (40 mg/m2/day x 4 doses), busulfan (3.2mg/kg/dose IV x 4 doses). Of these thirty patients, 7 had busulfan pharmacokinetics measured. There were 21 male and 9 female patients with a median age of 62 years (range 48–72yrs). Median time to follow up was 13.3 months. Diagnoses included AML (N=17), MDS (N=10), MPN (N=1), CMML (N=2). Disease risk was considered advanced in 17 patients, intermediate in 3 and early in 10. All patients in the busulfan pharmacokinetic group had advanced disease except one had early disease. Regarding cytogenetics, all patients in the busulfan pharmacokinetics group had high or intermediate risk cytogenetics. Median blast number at time of SCT was 5%. Stem cell source included peripheral blood in all patients. Eleven patients received matched related donor (MRD) SCT, 15 had matched unrelated donor (MUD) SCT and 4 had mismatched unrelated donor (MMUD) SCT. Prophylaxis of GVHD consisted of tacrolimus and methotrexate in MRD, and tacrolimus, methotrexate and anti-thymocyte globulin in MUD and MMUD.All patients engrafted neutrophils and platelets promptly (median 13 and 14 days, respectively). There were no primary graft failures. Total Donor cell Chimerism analysis in the Busulfan pharmacokinetics group showed 100% donor at both time points (days 30, 100) in all patients except in one who relapsed at day 30 (85.7%). While in the non-pharmacokinetics group only 7 out of 23 (30%) patients had complete chimerism at day 30 and day 100. Complete donor chimerism at day 100 in the non-pharmacokinetics group was 47.8% compared to 85.7% in the pharmacokinetic group (p=0.18). Ten out of 23 patients (43.5%) in the non-pharmacokinetics group had decreasing donor chimerism by day 100, while in the pharmacokinetic group only one patient (14%) who relapsed had decreasing donor chimerism by day 100 with an odds ratio of 0.241 (95% Confidence Interval =0.025-2.357; p-value=0.22). None developed sinusoidal obstructive syndrome.Conclusion: In this small cohort of consecutive patients from a single center, we found that patients with myeloid disorders who received fludarabine busulfan for 4 days incorporating busulfan pharmacokinetics had a trend for higher rates of early complete donor chimerism and less decreasing donor chimerism by day100, although not statistically significant, despite having intermediate or high risk disease at time of SCT. Longer follow up is needed for our patients to see if there is effect on relapse or survival but previous studies have showed that low or decreasing donor chimerism early after SCT is an independent risk factor for relapse and impaired survival. This is especially important in myeloid disorders. Busulfan pharmacokinetics may help target better level for inducing early total donor chimerism and donor chimerism may identify high-risk patient cohorts who may benefit from additional therapeutic interventions. These results should be interpreted with caution given the retrospective design of the study and the small number of patients. DisclosuresNo relevant conflicts of interest to declare.

Does HLA-C Matching Predict the Engrafting Unit in Double Umbilical Cord Blood Transplants?: A Single Institution Retrospective Analysis

▪Background: Lower total CD34+ cell dose and increased HLA-mismatch are known predictors of engraftment failure and higher transplant related mortality (TRM) in cord blood (CB) recipients. To compensate for cell dose, double unit grafts (DUCBT) are commonly used in adult. However, in the majority of patients (pts), only one of the two CB units engrafts. Identification of the factors that predict which unit will engraft remains elusive, although increased recipient-donor HLA-matching and larger CD3+ cell dose have been associated with the predominating unit in a single center retrospective analysis (Ramirez et al, 2012). Historically, CB units are selected by maximizing matching at the HLA-A and -B antigen and -DRB1 allelic level. Evidence supports that matching at HLA-C appears to decrease TRM, and many clinicians incorporate HLA-C antigen matching into unit selection. It is unclear, however, if HLA-C matching predicts the engrafting unit in DUCBT. This study retrospective study evaluates whether HLA-C matching is associated with the “winning” CB unit.Design: Clinical data was reviewed from all pts with a hematologic malignancy receiving a DUCBT at Moffitt Cancer Center between November 13, 2009 and August 29, 2013. Chimerism studies identified the predominating unit (> 65% single unit) between day 21 and day 28. Subsequent chimerism analyses performed at a median of day 100 confirmed unit predominance. Unit selection required intermediate resolution antigen match at HLA-A, -B, -C, and high resolution allele match at -DRB1. Units were a minimum of 4/8 matched to the patient and each other with a minimum cell dose of 1.5 x 107 total nucleated cell dose (TNC) /kg. Serology for donor specific antibodies against both units was negative.Results: Excluding 6 pts who were missing HLA-C typing on one or both CB units, 54 pts with hematologic malignancies (ALL=6, MDS/AML=29, Other=19) received chemotherapy and total body irradiation as part of a myeloablative conditioning (MAC) or reduced intensity conditioning (RIC) with or without thymoglobulin (ATG) followed by a DUCBT (MAC=14, RIC=23, RIC+ATG=17). Median age was 52 (range 22-69) years. Seven pts demonstrated persistent mixed chimerism in the myeloid and/or lymphoid cell lines beyond day 100. A total of 20 pts with available chimerism data received at least one CB unit matched to the recipient at HLA-C, with one patient excluded due to persistent mixed chimerism. Six pts received both CB units matched at HLA-C, but of the 13 pts receiving one matched and one mismatched unit, the HLA-C matched unit was the engrafting unit 69% (9/13) of the time. Comparing similar HLA mispairings, a matched unit engrafted over a mis-matched unit at HLA-A 50% (5/10) of the time, at HLA-B 38% (5/13) of the time, and at HLA-DRB1 50% (3/6) of the time. TNC dose (larger vs smaller with a required difference of at least 0.03 x 107 TNC/kg), order of infusion (first vs second unit), and overall CB unit HLA matching (4/8-8/8), were assessed as potential predictors for engraftment. Of evaluable patients, the CB unit with the larger TNC engrafted 44% (16/36) of the time, and the first unit infused was the engrafting unit 54% (21/39) of the time. In patients with an unequal overall match grade between the CB units, the better HLA-matched CB unit engrafted 64% (14/22) of the time. Survival analysis of all pts revealed that those who received at least one CB unit antigen matched at HLA-C (n=20) had a 100 day and 1 year overall survival (OS) of 85% (95% Confidence Interval(CI): 67–97%) and 55% (95% CI: 33–75), respectively, whereas pts receiving two HLA-C mismatched CB units (n=34), 100 day OS was 62% (95% CI: 45–77) with 1 year OS 44% (95% CI: 28–61) (Fig 1).Conclusion: HLA-C antigen matching appears to help predict the “winning” unit in settings of HLA-match disparity and DUCBT. Confirmation in a larger population of DUCBT recipients is necessary. To date, the effects of HLA matching and other variables influencing engraftment have been predominately evaluated in recipients of single unit transplants and studies in DUCBT have been limited. Further investigation assessing HLA matching as well as donor-donor interactions is best served through a multicenter data resource. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Persistence of recipient-derived as well as donor-derived clones of cytomegalovirus pp65-specific cytotoxic T cells long after allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus (CMV)-specific CD8(+) cytotoxic T lymphocytes (CMV-CTLs) play a crucial role in preventing CMV disease. However, the actual in vivo dynamics of CMV-CTL clones after allogeneic hematopoietic stem cell transplantation (alloHCT) are still unclear. Using a single-cell T-cell receptor repertoire analysis, we monitored clones and chimerism of CMV-CTLs in 3 CMV-seropositive alloHCT recipients from CMV-seronegative donors, with or without CMV reactivation. Nearly all of the CMV-CTLs during follow-up were CD45RA(-) CCR7(-) effector memory/CD45RA(+) CCR7(-) effector T cells, and were highly matured. In each case, the use of BV gene families was restricted, especially in BV5, 7, 28, and 29. Although no common predominant CMV-CTL clones were found, several shared motifs of complementarity-determining region-3 were identified among the 3 cases; QGA in all, TGE and TDT in Case 1 and Case 2, and RDRG in Case 2 and Case 3. In all cases, CMV-CTL clones that were detected for the first time after alloHCT persisted as the dominant clones. In Case 1, without CMV reactivation, recipient-derived CMV-CTLs exclusively persisted as a dominant clone, while all CMV-CTLs in the other 2 cases, with CMV reactivation, were donor derived. Clone monitoring and chimerism analyses should help to further clarify novel aspects of immuno-reconstitution after alloHCT.

Chimerism analysis in peripheral blood using indel quantitative real-time PCR is a useful tool to predict post-transplant relapse in acute leukemia.

Detection of increasing mixed chimerism (IMC) using standard PCR correlates with relapse after allo-SCT for acute leukemias (ALs). Quantitative real-time PCR of insertion/deletion polymorphism (indel qrtPCR) is a much more sensitive method, which can be performed on peripheral blood. We studied the significance of low increases of recipient cells (0.1%) detected by indel qrtPCR in a cohort of 89 transplants. We did not observe relapse among the 32 patients with no IMC. Fifty-seven patients presented a first IMC, which was followed by four different scenarios: a decreasing MC (26 cases, no relapse), a stable MC (1 case, 1 relapse), a second IMC (24 cases, 15 relapse) or no control of chimerism (6 cases, 5 relapses). In multivariate analysis, detection of two successive IMCs was strongly associated with relapse (hazard ratio (HR): 9.4, 95% confidence interval (CI): 3.8-23; P<0.0001). Among the 57 patients who presented at least one IMC, 27 underwent immunomodulation (tapering of immunosuppression or donor lymphocyte injection), leading to a 1-year relapse rate of 15.7% vs 57.6% in the 30 other patients (P=0.0007). Altogether, these results indicate that chimerism analysis using indel qrtPCR in peripheral blood is a useful tool for detection of relapse in patients transplanted for AL.