Chimerism Status Research Articles

Cell-free DNA characteristics and chimerism analysis in patients after allogeneic cell transplantation

Cell-free DNA (cfDNA) isolated from plasma or serum has received increasing interest for diagnostic applications in pregnancy, solid tumors and solid organ transplantation. The reported clinical usefulness of cfDNA obtained from plasma or serum in patients undergoing allogeneic cell transplantation (alloHSCT) is scarce. ObjectiveTo analyze the potential clinical utility of cfDNA chimerism analysis after alloHSCT. Design and methodsA total of 196 samples obtained from 110 patients were investigated for their chimeric status both in peripheral blood and plasma using standard PCR for microsatellite amplification. Plasma DNA size distribution was analyzed using capillary electrophoresis. ResultsThe mean cfDNA concentration in the transplanted patients was 469ng/ml (range: 50–10,700ng/ml). The size range of almost 80% of the analyzed fragments was between 80 and 200bp. In 41 out of the 110 patients included in the study a mixture of donor and recipient plasma cfDNA was detected. There was a statistically significant difference in the percentage of plasma mixed chimerism between the patients without transplant related complications and the patients with either GvHD (p<0.05) or relapse (p<0.01). In those patients who showed improvement of GvHD also displayed a decrease in the observable percentage of recipient cfDNA during GvHD treatment. In patients without improvement or even with worsening of acute GvHD, stable or increasing levels of recipient cfDNA were detected. ConclusionscfDNA in combination with peripheral blood and bone marrow cell chimerism analysis might improve its utility in the clinic in particular in those patients with clinical complications after alloHSCT.

P070 A post-transplant chimerism study protocol that incoporated both STR and QPCR methods

Aim To monitor post-stem cell transplant chimerism status, most clinical laboratories use commercial kits for short tandem repeat (STR) or quantitative real-time PCR (qPCR). We present a protocol that combines both systems to maximize monitoring performance. Methods PowerPlex Fusion STR kit (Promega) was used to genotype 22 autosomal STR markers. Donor (%) was assigned as an average from five informative loci (Types 1 and 2). Fragment analysis used 3130xl Genetic Analyzer (Applied Biosystems) and GeneMapper software (Thermo Fisher Scientific). KMRType kit (GenDx) was used for genotyping 29 insertion/deletion markers (4-color multiplex) on QuantStudio 12K Flex Real-Time PCR instrument (Thermo Fisher). Two informative loci (insertion in recipient -deletion in donor) selected were used to determine minor allele (%) (one-color) by KMRTrack kit (GenDx) on the Roche LightCycler480 Real-Time PCR instrument (KMRTrack). The average from the two loci was assigned as minor allele (%). Results The average number of STR informative loci for a patient-donor pair was 18.3 (range: 16–21) for unrelated (N = 30) and 9.0 (range: 6–16) for related (N = 30). The number of qPCR informative loci was 6.0 (range: 4–9) for unrelated (N = 28) and 3.5 (range: 3–4) for related (N = 2). In qPCR, sensitivity increases when more DNA is used per reaction and surpasses STR sensitivity of 3–5% when >2 ng DNA is used per reaction. We validated the qPCR method using 183 different donor-recipient-marker combinations and 14 ng whole blood DNA/reaction, which achieved sensitivity of 0.5%. Correlation coefficient (r) of intended % donor (mixed DNA) vs. assay-determined was 0.999 by STR (N = 12 for range: 5–95% donor) and 0.91 by qPCR (range: 0.5–10% minor allele). Average %CV was similar at low minor allele range; STR (29.6% for 5% donor detection) vs. qPCR (average 27.8% for detecting 0.5%–10% minor allele). At high minor allele range, %CV decreased for STR (CV Conclusions The combination of STR and qPCR can improve a post-transplant monitoring protocol. STR (higher precision) is used immediately after transplantation when high recipient (minor) alleles are present. Testing will be switched to qPCR (higher sensitivity) once complete chimerism is achieved.

P035 Evaluation of a marker set for chimerism monitoring by next-generation sequencing

Accurate monitoring of the chimeric status of patients after stem cell transplantation is essential for early detection of transplant rejection or relapse. Currently, micro-chimerism levels are mostly assessed by Short Tandem Repeat (STR) fragment length analysis on capillary electrophoresis systems. The major disadvantages of this technique are the poor, low-level sensitivity and the laborious data analysis. As more and more HLA-typing labs have access to NGS, it is efficient to also use NGS for chimerism monitoring. We hypothesize that NGS-based chimerism monitoring will have a higher sensitivity than conventional methods and have a wider dynamic range, also including the dynamic range of STR technology. Here, we present the preliminary results of chimerism testing by means of NGS and evaluate the use of different types of informative markers. PCR amplification primers have been designed for a set of polymorphic InDel and STR markers. Using these markers a number of artificial chimeric samples were amplified. NGS data was analysed with customized analysis tools designed to quantitate the absence, presence, and length of the markers. Non-chimeric samples tested result in clear homo- or heterozygous patterns for all markers tested. No interfering noise levels were found higher than 0.05% of the total number of reads assigned to that amplicon. When conducting artificial chimerism tests in the range between 0.1% and 100%, all percentages included could clearly be differentiated. Correlation between allele ratio targeted and percentages found exceeded R2 = 0.995 for a number of markers. The results demonstrate that NGS-based chimerism monitoring is feasible, with a high sensitivity and wide dynamic range, for multiple markers tested.

Early mixed chimerism-based preemptive immunotherapy in children undergoing allogeneic hematopoietic stem cell transplantation for acute leukemia.

This retrospective analysis comprises 10-year experience with early posttransplant mixed chimerism-based preemptive intervention. Out of 104 patients, 51 received preemptive immunotherapy. Their outcomes were similar to patients achieving full donor chimerism spontaneously. Among patients receiving intervention, 5-year event-free survival was identical in patients with and without pretransplant residual disease, respectively (68% [95% confidence interval (CI) 38-98%] vs. 69% [95% CI 54-85%] log-rank = 0.4). In patients who received preemptive immunotherapy, chimerism status and residual disease prior to transplant were no longer predictors of poor outcome; however, 41% of the patients with residual disease prior to transplant relapsed early and did not benefit from this strategy.

Dose-adapted post-transplant cyclophosphamide for HLA-haploidentical transplantation in Fanconi anemia.

We developed a haploidentical transplantation protocol with post-transplant cyclophosphamide (CY) for in vivo T-cell depletion using a novel adapted-dosing schedule (25 mg/kg on days +3 and +4) for Fanconi Anemia. With median follow-up of 3 years (range, 37 days to 6.2 years), all six patients engrafted. Two patients with multiple co-morbidities and late referrals to transplant died from sepsis (n=2) and chronic graft-versus-host disease (GVHD) (n=1). Four patients without pre-existing co-morbidities and early transplant referrals are alive with 100% donor chimerism and excellent performance status. We conclude that modulated-dosing post-transplant CY is effective in vivo T-cell depletion to promote full donor engraftment in patients with Fanconi anemia.

Novel deletion mutation of HLA-B*40:02 gene in acquired aplastic anemia.

Despite prevalence of clonal evolution in patients with aplastic anemia (AA), somatic mutation of human leukocyte antigen (HLA) gene is rarely reported. Herein, we reported a case of acquired AA (aAA) harboring a new four-base-pair deletion mutation within exon 4 of HLA-B*40:02 leading to frameshift and premature stop codon. The HLA-B*40:02 mutant allele was detected in the patient's peripheral blood sample not in patient's buccal epithelial cells. The patient received allogenic hematopoietic stem cell transplantation (HSCT) from HLA-matched sibling donor. On day 30 after HSCT, the mutant HLA allele was not detected by high-resolution sequence-based HLA typing. Serial chimerism analyses showed mixed chimeric status indicative of coexisting donor and recipient hematopoietic cells. Our data could provide additional support in view of pathophysiology of aAA that somatic mutation of HLA-B*40:02 allele is one of the possible origin of clonal escape to evade immune attack in patient with aAA.

Engraftment of Donor Cells after Allogeneic Stem Cell Transplantation: Comparison and Impact of Chimerism in Whole Blood and Peripheral CD3+ T-Cells

Introduction: After allogeneic stem cell transplantation (allo-SCT), engraftment can be assessed by quantitative polymerase chain reaction (qPCR) using differing donor/recipient markers (Alizadeh et al. Blood, 2002), identified in peripheral blood DNA cells before transplant. We report here on the concomitant examination of the proportions of donor and recipient DNA in peripheral whole blood (WB) and sorted CD3+ T-cells on days +60 and +90, looking at their impact on survival.Patients, material and methods: This monocentric study evaluated the impact on outcomes of early WB and sorted CD3+ T cells chimerism independently and of the four possible combinations of chimerism between WB and sorted CD3+ T-cells. All follow-up chimerism samples from allo-SCT patients performed in adults at Nantes University Hospital between October 2009 and October 2016 were reviewed, focusing on those where both PB and/or CD3+ T-cells were evaluated on days +60 (45-75) and/or +90 (75-120) after allo-SCT. A global cohort of 229 patients (239 grafts) was retrieved, which includes 52 patients evaluable on day +60 only, 67 evaluable on day +90 only and 120 evaluable on both days +60 and +90. A threshold of >95% donor DNA was considered for complete chimerism. Disease free survival (DFS) was calculated from the date of graft until relapse, death or last follow-up. Overall survival (OS) was calculated from the date of graft until death or last follow-up. Chi square tests were used to compare incidences. Log rang test and Kaplan Meier were used to evaluate DFS and OS.Results: The whole cohort comprised 62% males and had a median age of 58 years old (20-74) at the time of allo-SCT. Patients were treated for myeloid-lineage disease in 59% of the cases. Reduced-intensity conditioning was used in 89% (n=212), donors were familial in 45% (n=107), registry in 48% (n=114). Unrelated cord blood units were used in 8% of the cases (n=18). Post-transplant cyclophosphamide (PTCY) was performed in 48 procedures including 33 and 15 with haplo (HG) and matched donors respectively. Considering the 239 allograft procedures, the median follow-up was 5.8 years (95% CI: 3.1-5.8), the rate of relapse 27% and the rate of death 31%.Complete WB chimerism was observed for 80% and 71% of the cases on day +60 and day +90 respectively. Complete CD3+ chimerism was present for 53% and 51% of the grafts at days +30 and +90 respectively. Thus, cases displaying both complete WB and CD3+ chimerism on days +60 and +90 were 53% and 51% respectively, while 27% and 20% were documented with full WB and mixed CD3+ chimerism on days +60 and +90. Mixed chimerism was observed in both WB and CD3+ cells in 14% of the cases on day +60 and 22% on day +90. Finally, a small proportion of patients (6% and 7% at days +60 and +90) displayed an intriguing complete chimerism in CD3+ cells yet mixed WB chimerism. None of these features appeared associated to disease lineage (lymphoid or myeloid) nor cord blood allo-SCT. Interestingly, of the 27 grafts with myeloablative conditioning, only 14 had full WB/CD3+ engraftment on day +60 or +90, and thus all 27 were retained for the study.None of the four WB/CD3 chimerism combinations at the two times considered had an impact on DFS in this cohort. Surprisingly, although full or mixed WB chimerism had no impact on DFS and OS at days +60 and +90, the presence of a mixed CD3+ chimerism (vs full) at day+90 was associated with a significantly better OS (median: 5.8 months years [95%CI: -not reached] versus 3.1 years [95%CI: 2.2- 3.1]; p=0.025). CD3+ chimerism at day+60 has no impact on OS.All HG resulted in full CD3+ chimerism at both time points compared to non HG (100% vs 52%, p<0.0001). The same was almost true when considering PTCY procedures: 90% at day+60 and 92% at day +90. Of note, there was no influence on DFS nor OS of WB or CD3+ chimerism status when considering only HG or PTCY grafts vs others in this series.Discussion: In this large series, early WB chimerism status did not predict outcome. Surprisingly, mixed CD3+ chimerism at day+90 appears to be significantly associated with a longer OS, suggesting that remaining recipient memory lymphocytes could be beneficial. This result has to be confirmed prospectively. It remains also to define the place of donor lymphocyte infusions (DLI) to prevent relapse in patients with full or mixed CD3+ chimerism post-transplant (analyses of DLI received in our patients are on-going). DisclosuresMoreau:Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria.

Long Term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation for Beta Thalassemia Major

▪Introduction:Allogeneic hematopoietic stem cell transplantation (HSCT) cures beta thalassemia major (TM). Such individuals, ex-thalassemics, have good long term survival. However, there is limited data on long term outcome (LTO) of this therapy. This is particularly relevant as these patients often have organ dysfunction pre-transplant due to secondary hemosiderosis apart from the impact of post-transplant factors such as chronic GVHD, chimerism status and iron depletion therapy (IDT). In this report, we describe the LTO of patients with TM who underwent HSCT with busulfan (Bu) and cyclophosphamide (Cy) conditioning at our center from 2000 to 2011 and had a minimum of 2 year follow-up.Method: Data was extracted from prospectively maintained standardized case record forms for details of HSCT and long term follow-up with particular reference to GVHD, chimerism (evaluated at day +30, +60, +100 and thereafter as indicated), IDT (initiated at variable periods post-HSCT) and metabolic and endocrine disorders evaluated on physician discretion or as per clinical indications.Results:A total of 190 patients underwent matched related donor HSCT from 2001 to 2011 with Bu/Cy based conditioning. After excluding those who expired or had primary graft failure or did not have at least 2 years of follow-up, 124 patients were available for analysis of LTO. 44 patients (35.5%) class 3, 69 patients (55.6%) class 2 and 11 patients (8.9%) class 1. The median age was 7 years (range: 2-24) with 81 males (65.3%). The median follow-up was 7 years (range: 2 to 14). Chronic GVHD was present in 22 patients (17.7%]. Mixed chimerism (MC) occurred in 40 patients (32%) in the first year after HSCT: level I in 21 (52.5%), level II in 10 (25%), level III in 7 (17.5%), and level unknown in 2(5%). At last follow-up, 20/40 (50%) patients with MC went on to CC, 18 maintained stable MC (level I-5, level II-9 and level III-4) with hemoglobin of 11.35g/dl (range: 9-13.5), while 2 (5%) with level 3 MC remained transfusion dependent. Median serum ferritin (SF) at HSCT was 2367 ng/ml (range: 685-7660). IDT was initiated in 90 (72.6%) patients at a median of 15 months (range: 6-53) post-HSCT - 13 patients (14.4%) were treated with phlebotomy alone, while 39 (43.3%) received chelation and 38 (42.2%) the combination. Reduction in SF/month [absolute quantity (ng/ml/month) and percent] was as follows: 40.5 (range: 11.68 - 125.78); 1.67% (range: 0.5-4.58), 54.9 (range: 9.3- 278.7); 2.1% (range: 0.41- 13.8) and 36.6 (range: 3.51-590.7); 1.3% (range: 0.42-42.99), in the phlebotomy, chelation and combination groups, (p=0.077 & 0.017, respectively). SF level of <300 ng/ml was achieved in 33 patients (31%) at last follow-up. Anthropometry measurements (at last follow up) revealed short stature in 53 patients (42.7%; 38M/15F), underweight in 32 patients (25.8%; 20M/12F) and overweight in 14 (11.3%) patients (11M/3F). A total 48 patients (38.7%) had the following endocrine disorders: hypogonadism in 33 (73.3%), primary hypothyroidism in 9 (18.8%), hypopituitarism in 4 (8.3%), diabetes mellitus in 3 (6.2%), and hypoparathyroidism, dyslipidemia and hypertension in 1 patients each. 40 patients were vitamin D deficient (83.3%). Endocrine complications were more common in female patients (55.8% versus 29.6%; p=0.006). Two patients (1.3%) developed malignancies at 7 and 8 years, post-HSCT. Among different patient, donor and graft characteristics, there were no predictors of MC, nor did the ferritin levels or chelation therapy post-HSCT affect the incidence of endocrine complications in this cohort.Conclusion: Our data shows that even though the long term survival of ex-thalassemics is extremely good, at least 40% of them suffer from several co-morbidities related to iron overload and various metabolic and endocrine disorders which requires a coordinated plan for their management. The aim therefore should be to transplant these patients as early as possible before such complications occur and implement IDT intensively early after HSCT. DisclosuresNo relevant conflicts of interest to declare.

Complementary Transplantation with Haploidentical Stem Cells and Unrelated Cord Blood for Leukemia and Thalassemia

BackgroundUnrelated Cord Blood (UCB) Transplantation is a potentially curative therapy for leukemia and thalassemia; however, engraftment failure and slow immune reconstitution remain key clinical issues. We hypothesized that complementary transplantation (CT) of UCB with haploidentical stem cell graft (hap-SC) tolerized with post-transplant cyclophosphamide would result in rapid engraftment and low relapse rate without additional risk of graft-versus-host disease (GVHD). Therefore, we developed a novel complementary transplant approach.Patients and MethodSixty-six patients received CT between December 2012 and June 2016. Of them, 30 patients had malignance diseases (MD), including 11 lymphoid and 19 myeloid diseases, and 36 had thalassemia major (TM). Median age was 12 (range; 2-13) and 8 (3-17) years in the MD and TM group, respectively. Median follow-up time was 13 (7-32) and 19 (2-25) months, respectively. Conditioning (Regimen CT-13) included Cyclophosphamide on day-8 to -7, Busulfan on day-6 to -4, Fludarabine on day-6 to -2 and Thiotepa on day-3. Hap-SC was infused on day 0. GVHD prophylaxis consisted of Cyclophosphamide on day+3 to +4. UCB was infused on Day+6. Mycophenolate mofetil and Tacrolimuswas started on day+6 for GVHD prophylaxis. For 26 TM patients transplanted since 2014, they received identical regimen except with the additional Thymoglobulin on day -11 to -9 (Regimen CT-14).ResultsThe chimerism status at last follow-up was Hap-SC, UCB, mixed stem cells (MSC) and rejection in 20, 9, 1 and 0 patient in the MD group; and 16, 14, 3 and 3 patients in the TM group. Interesting, the initial chimerism on day+28 in the TM group was Hap-SC, UCB and MSC engrafted in 15, 7 and 12 patients, respectively. Thus, the MSC was not stable in TM patients; UCB typically became dominant overtime instead of the initial majority from hap-SC (Fig. 1).In the MD group, the time to neutrophil >= 0.5x109/L, platelet >=20 x109/L and hemoglobin >=80 g/L was day+18 (14-36), +10.0 (6-51) and +7 (1-20) in the final hap-SC engrafted group; and+30 (22-35), +25 (1-64) and +7 (3-28) in the final UCB engrafted group. Overall survive (OS), disease-free survive (DFS), relapse incidence (RI) and non-relapse mortality (NRM) were 75.6%, 64.3%, 24.7£¥ and 13.7%, respectively, in all 30 MD patients; and 79.3%,73.8%,15.5% and 12.3% (Fig.2), respectively, in the 25 cases with complete remission (CR) at the time of transplantation. The corresponding data were 89% vs. 88.9%£¬65.0% vs. 77.8%£¬31.1 % vs.12.5% and 10.3% vs.11.1%£¨p>0.05 in all pairs), respectively, in hap-SC and UCB engrafted groups. Donor carrying KIR centromeric B motif was associated with reduced RI (10 % vs. 33.9%).In TM group, OS, thalassemia free survive (TFS), rejection and transplant-related mortality were 91.2%, 85.7%, 5.6% and 8.8%, respectively in all 36 patients. Impressively, all of the 26 patients who received the newer CT-14 protocol were alive without TM (Fig. 2). 7/10 UCB carrying KIR centromeric B motif engrafted.In the MD group, 23.3% had grade II-IV and10.0% had III-IV acute GVHD. Grade II, III and IV acute GVHD occurred in 3 patients, respectively, in TM group. One MD patient had severe chronic GVHD (lung) after DLI for relapse. No moderate chronic GVHD occurred in TM groups.SummaryThe CT-13 regimen resulted in high OS and DFS, especially in CR patients in the MD group. The CT-14 leaded to 100% TFS in thalassemia patients. Acute and chronic GVHD were acceptable. Donor carrying centromeric B motif promoted engraftment and reduced RI. A multicenter study should be developed in the future based on our favorable results. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Can Memory T Cell Monitoring Using Flow Cytometry Predict Survival 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. One of the advantages of HHSCT is the possibility to use donor cells for post-transplantation cell therapy. However, HHSCT has risks associated with HLA barrier, such as graft failure, severe graft-versus-host disease (GVHD), and delayed immune reconstitution. T cell receptor gamma delta (TCRγδ) T cells may have potent antileukemic effects. These cells can be preserved in a graft by negatively selecting only T cells that express an alpha beta (αβ) T cell receptor (TCR αβ). Moreover, γδ T cells are important effector cells, especially in situations where the function of adaptive immunity is impaired, such as those characterizing early immune recovery after HHSCT. In this study, we performed flow cytometry (FCM)-based T cell and TCRγδ memory T cell subpopulation analysis with anti-HLA antibodies to monitor the changes of memory T cell subpopulations after HHSCT according to clinical course.Methods:Peripheral blood samples of total eighty-one pediatric patients who underwent HHSCT in Asan Medical Center were collected between October 2011 and June 2016. Diagnoses were aplastic anemia (n=31), acute myeloid leukemia (n=20), acute lymphoblastic leukemia (n=15), non-Hodgkin’s lymphoma (n=10), non-malignant hematologic disorders (n=5). Four patients received CD34-selected graft, 26 patients received CD3-depleted graft, and 51 patients received TCRαß-depleted graft. Patients who experienced graft loss or disease relapse were fifteen. Seventeen patients received zoledronate (Zol) and interleukin-2 (IL-2) to augment TCRγδ T cells after HHSCT. Twelve patients were expired. Nineteen patients were experienced GVHD. FCM analysis was performed using antibodies for HLA antigens and anti-CD45, CD3, CD4, CD8, CD45RA, CD45RO, CD62L and TCRγδ antibody to identify naïve, central memory (CM) and effector memory (EM) T cells. The anti-HLA antibody can be used to evaluate chimeric status in HHSCT based on disparity of HLA antigens between donor and recipient. FCM analysis was done according to regularly scheduled protocol from the start of stem cell infusion. We evaluated the differences of T cell and memory T cell subpopulations and Treg according to engraftment status, treatment strategy, survival status, GVHD status, and the types of underlying disease were analyzed.Results: At the early stage (0-30 day) after HHSCT, the absolute counts of TCRγδ CM, EM and naïve T cells were higher in engraftment group than graft failure (CM 2.71/µL vs. 0.19/µL, EM 0.63 vs. 0.00, and Naïve 2.50 vs. 0.37, P<0.05). The administration of Zol+IL-2 significantly increases TCRγδ memory cell absolute counts (CM 4.51/µL vs. 0.68/µL, EM 1.78 vs. 0.00, and Naïve 2.73 vs. 0.72, P<0.05) during early period after HHSCT. These drugs also resulted in higher donor T cell count (147.56 vs. 60.76, P=0.025) and donor TCRγδ T cell count (92.96 vs. 43.9, P=0.046). After 6 months of HHSCT, survived patients showed lower percentage of EM cells than non-survivors (CD4+EM 5.69% vs. 22.22%, and CD8+EM 11.13 vs. 66.79, P<0.05), and higher percentage of CM cells (CD4+CM 59.11% vs. 36.84%, P=0.02, and CD8+CM 29.73% vs. 1.83%, P=0.00) than expired patients. Patients with GVHD showed significantly lower percentage of Naïve T cells than patients without GVHD (CD4+Naïve 0.00% vs. 68.64%, and CD8+Naïve 1.77 vs. 41.31, P<0.05). The percentage of TCRγδ T cell was higher in malignant disease patients than non-malignant disease patients (15.61 vs. 4.75, P=0.008).Conclusions: The increase of TCRγδ memory cells in the early period after HHSCT could expect engraftment. The administration of Zol+IL-2 augmented TCRγδ memory cells during early period after HHSCT. Therefore, these drugs might be related with engraftment maintenance. The monitoring of reconstitution pattern of central and effector memory T cell after 6 months of HHSCT could predict survival. Our study suggests that close monitoring of FCM-based memory T cell subpopulation is useful to predict clinical outcome after HHSCT. DisclosuresNo relevant conflicts of interest to declare.

A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

P021 Development of laboratory-developed chimerism analysis assay targeting t lymphocytes in hematopoietic stem cell transplantation

Aim Hematopoietic stem cells transplantation (HSCT) is one of the gold standard treatment for the patients with hematologic malignancies. After the transplantations, the engraftment of the stem cells is monitored using various methods. Short tandem repeats (STR) are one of the sensitive markers for chimerism. We designed laboratory-developed chimerism analysis assay using 8 short tandem repeat regions. Methods Total 38 patients who received allogeneic HSCT were involved in this study. The diagnosis of the patients included acute myeloid leukemia, precursor lymphoblastic leukemia, myelodysplastic syndrome, hemophagocytic lymphohistiocytosis, chronic eosinophilic leukemia, paroxysmal nocturnal hemoglobinuria, juvenile myelomonocytic leukemia, plasma cell myeloma, and congenital neutropenia. DNA was extracted from both ACD anticoagulated blood and T lymphocytes isolated by EasySep (STEMCELL Technologies, Vancouver, Canada). We designed the assays, named LDT 8, targeting D3S1358, D5S818, TH01, D7S820, D12S391, FGA, D18S51, and Penta E. The amplicons were analyzed using Performance Optimized Polymer 7 (Applied Biosystems Norwalk, USA) by 3130 xl genetic analyzer (Applied Biosystems). The results LDT8 were compared with commercially available PowerPlex 21 (Promega, Madison, USA) and Mentype 13 (Biotype, Dresden, Germany). In addition, we also compared the chimerism status of whole blood to that of T lymphocyte. Results Compared with PowerPlex 21 and Mentype 13 respectively, 90.1% and 84.9% of specimens showed less than 5%p in overall discrepancies. In the comparison between whole blood and T lymphocytes, most of specimens showed the higher recipient proportion of DNA in T lymphocytes. Conclusions We developed the economical and effective chimerism analysis assay using 8 short tandem repeat regions. Also, we identified discrepancies among two different cell sources. Further study for long-term monitoring of chimerism status in different cell subsets would be helpful in patients who received hematopoietic stem cell transplantation.

State of Chimerism as a Promising Tool for Follow up of Hematopoietic Stem Cell Transplantation in Egyptian Patients

Follow up of patients after HSCT by chimerism detection can detect the outcome either engraftment, relapse or rejection. Many variables affect the result of chimerism either increasing or decreasing chimerism. Lineage specific chimerism is specific and sensitive than chimerism detection by whole blood. Here in this study the importance of these variables on the outcome of chimerism detection is studied. Subject and method: This study was conducted at Ain Shams University Hospital during the periodbetween January 2011and January 2015. 46 patients were included (16 child and 30 adult). They weretransplanted for malignant and non malignant hematological diseases from totally matched siblings.Themedian duration of follow-up was one year. Chimerism status was detected for all patients by polymerase chain reaction based on amplification of variable number tandem repeat (VNTR) markers. Six VNTR loci were detected in every subject pre-transplant in order to detect an informative locus to be used in follow uppost-transplant. Also lineage specific chimerism and dilution experiment were conducted on some patients. Results: One patient showed no informative locus pre-transplantation, 37 patients showed complete donor chimersm, two patients retained recipient pattern post-transplantation and six patients showed MC. Lineage cell specific chimerism and dilution experiment were conducted for patients who showed failure or mixed chimerism. Conclusion: Chimerism detection by VNTR is dependable method to follow up patients after BMT however,some cases need some modifications as repeated analysis, short duration, lineage specific in comparison to whole blood analysis, dilution experiments in order to predict the outcome.

Relapse of AML after hematopoietic stem cell transplantation: methods of monitoring and preventive strategies. A review from the ALWP of the EBMT.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) remains the therapeutic method with the most potent anti-leukemic activity mediated by the graft versus leukemia effect. However, a significant proportion of patients with AML will relapse after allo-SCT. The prognosis for these patients is dismal, with a probability of long-term survival of <20%. Data from previous studies have shown that disease-specific prognostic factors, are in general, the same as those in patients treated with conventional chemotherapy. Minimal residual disease (MRD) and chimerism status monitoring after allo-SCT may be used as predictors of impending relapse and should be part of routine follow-up for AML patients. A significant number of studies have shown that pre-emptive administration of donor lymphocyte infusion (DLI) based on MRD and chimerism monitoring, as well as prophylactic DLI in AML patients at high risk of relapse is effective in preventing relapse. In this review, we discuss strategies for the identification of high-risk patients, review current therapeutic options and provide our recommendations for the management of post-SCT AML.

Peripheral blood late mixed chimerism in leucocyte subpopulations following allogeneic stem cell transplantation for childhood malignancies: does it matter?

The impact of persistent mixed chimerism (MC) after haematopoietic stem cell transplantation (HSCT) remains unclarified. We investigated the incidence of MC in peripheral blood beyond day +50 after HSCT and its impact on rejection, chronic graft-versus-host disease (c-GvHD) and relapse in 161 children receiving allogeneic HSCT for haematological malignancies. The 1-year incidence of late MC was 26%. Spontaneous conversion to complete donor chimerism (CC) occurred in 43% of patients as compared to 62% after donor lymphocyte infusions. No graft rejection occurred. The 1-year incidence of c-GvHD was 20±7% for MC, and 18±4% for CC patients (P=0·734). The 3-year cumulative incidence of relapse (CIR) according to chimerism status at days +50 and +100 was 22±4% for CC patients vs. 22±8% for MC patients (day +50; P=0·935) and 21±4% vs. 20±7% (day +100; P=0·907). Three-year CIRs in patients with persistent MC and patients with CC/limited MC were comparable (8±7% vs. 19±4%; P=0·960). HSCT for acute leukaemia or myelodysplastic syndrome as secondary malignancies (hazard ratio (HR) 4·7; P=0·008), for AML (HR 3·0; P=0·02) and from mismatched donors (HR 3·1; P=0·03) were independent factors associated with relapse. Our data suggest that late MC neither protects from c-GvHD nor does it reliably predict impending disease relapse.

The Development of Methods for High Purity Hematopoietic Lineage Cell Isolation and Lineage Chimerism Testing

Chimerism analysis has become an important tool for assessing the origin of hematopoietic cells after allogeneic donor stem cell transplantations. Particularly, lineage chimerism analysis provides crucial information regarding treatment progress. Donor chimerism status for certain lineages, such as T, NK and myeloid, during early stages following post-transplant is associated with long term engraftment success and the relapse risk. However, the accurate lineage chimerism result is highly contingent on obtaining lineage cell preparation purity. There are several challenges in the field relating to the isolation of high purity cell lineages, chiefly among which is extremely low cell counts during the early stages post-transplant. Our laboratory has developed a method based on a) sequential isolation of hematopoietic lineages; b) purity assessment by flow cytometry; and c) a correction algorithm substandard purity of isolated cells. First, myeloid, B, T, and NK cells are sequentially isolated by magnetic separation using lineage specific markers. Thus each lineage isolated is largely excluded from the sample as a potential contaminant in each subsequent step. A purity with >90% can be routinely achieved when isolating lineages accounting for as little as 0.5 % of total leukocytes. Second, the purity of each population is verified by Flow Cytometry using a 6-color antibody panel staining for T, B, NK, myeloid, and total leukocytes. This enables not only the determination of cell purity of each isolated lineage cells, but also the identification and quantification of any contaminating cells in each isolated lineage. Lastly, an algorithm was derived based on the assumption that total chimerism consists of the four main lineages, T, B, NK, and myeloid. This mathematical correction is reflexively applied when isolated lineage cells are of <90% purity, often occurred as the isolated lineage accounting for < 0.5% of total leukocytes in the sample. In summary, our methods enable generation of high purity of isolated lineage cells that lead to accurate and reliable single lineage chimerism results.

Population Pharmacokinetics of Fludarabine and Treosulfan in Patients with Thalassemia Undergoing Hematopoietic Stem Cell Transplantation

A toxicity reduced conditioning regimen containing Treosulfan (Treo), fludarabine (Flu), thiotepa for high risk Thal Major (TM) has been used since 2009 at our centre that has significantly improved transplant outcomes of these patients compared to the historical cohort of patients receiving busulfan/ cyclophosphamide based myeloablative regimen (Mathews et al, 2013). Limited knowledge is available on the pharmacokinetics (PK), pharmacogenetics (PG) and pharmacodynamics of fludarabine and treosulfan, especially in non-malignant hematological disorders like TM. We describe here the PK of Flu and Treo in patients with TM undergoing HSCT, the factors influencing the inter-individual variability in PK and the role of these factors on HSCT outcome.Seventy one patients diagnosed with TM undergoing HSCT with Flu/Treo based conditioning regimen between January 2012 and January 2015 were included (Table: Patient demographics). Selected functional polymorphisms in the NT5E, DCK, hENT1 and GST genes that are involved in fludarabine or treosulfan metabolism were screened. All patients received Flu 40mg/m2/day x 4 days as an 1hr infusion on days 1 and 4 and Treo as 14g/m2/day x 3 days at the rate 5g/hr. Plasma was separated from the peripheral blood collected at predetermined time points after the infusion of Flu and Treo PK analysis. Plasma Flu was analyzed using a LC-MS/MS method and the concentration was expressed as mMole/ml while Treo was analyzed using a HPLC-RI method and concentration was expressed as mg/L. Flu and Treo PK was estimated using nonlinear mixed effects modeling via Monolix 4.3.3. The covariates tested for both PK were: age, sex, body weight, BSA, ferritin, and polymorphisms in NT5E, hENT1, dCK and GST genes. The PK parameters AUC, CL, V and k were estimated on day 1 for Treo and on day 1 and day 4 for Flu (Table). The influence of Flu and Treo PK and PG on graft rejection, early transplant related mortality (TRM) & chimerism status was estimated using logistic regression analysis.Wide inter-individual variation in Flu and Treo PK was noted (7 and 9 fold Vs 5 and 8 fold respectively for Day 1 & 4 Flu AUC & Cl; 33 & 31 fold variation in Treo AUC and Cl) (Table). Flu CL was significantly higher on day 4 compared to day1 (Figure A). The variation in Flu PK was explained by genetic variants in NT5E and dCK. Patients having variant genotype for the SNPs in NT5E (rs2295890) and dCK (rs11544786) showed significantly lower plasma Flu clearance compared to those with wild type genotype (p=0.006 & p=0.05 respectively) (Figure B). This is consistent with our previous report in patients with aplastic anemia undergoing HSCT (Mohanan et al. 2014; Blood: 124 (21)). None of the genetic variants in the GST genes explained the variation in Treo PK.Day21 mortality was seen in 6/71 patients (8.5%) and graft rejection in 3/66 evaluable patients (4.5%). Analysis of the influence of PK and PG variables on transplant outcome showed significantly high first dose Flu AUC to be associated with D21 mortality upon Univariate analysis (median 42.5, range 32.1-63.7 compared to 31.8, range 15.2-111 mMole*h/mL, in those with and without TRM respectively; p=0.043); none of these parameters were significantly associated with graft rejection or mixed chimerism. There was no association between Treo PK parameters and graft rejection or TRM. The influence of Flu and Treo PK on regimen related toxicity is yet to be evaluated. The lack of the influence of PK on transplant outcome could be due to lower incidence of rejection and TRM in this cohort. Further analysis in a larger cohort of patients will be done once we enroll more patients for PK analysis.Our results demonstrate that Flu PK is influenced by genetic variants in NT5E and dCK, the enzymes involved in Flu biotransformation. The relationship between high-plasma Flu exposure and TRM and given the fact that multiple factors influence TRM, we can extrapolate that the plasma Flu AUC may be a surrogate marker of overall preparative regimen intensity as reported previously (Long-Boyle et al, Bone Marrow Transplant, 2011). The lack of association of genetic variants in GST genes in explaining the inter-patient variability in treosulfan exposure suggests the involvement of other drug metabolizing genes on treosulfan PK. We are currently evaluating the role of genetic variants in a large panel of drug metabolizing genes on explaining this inter-individual variability in Treo PK. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytomegalovirus and Effect on Early Chimerism in Patients with Myeloid Disorders Undergoing Stem Cell Transplantation Using Reduced Toxicity Ablative Conditioning Regimen

Cytomegalovirus (CMV) remains a significant cause of morbidity after allogeneic hematopoietic stem cell transplantation (SCT). The influence of CMV on the chimerism in reduced toxicity ablative conditioning SCT in myeloid disorders is ill defined. A recent report published in Blood by Sellar et al showed that in patients who received alemtuzumab-based regimen, the group of patients who were recipient positive (R+)/ Donor negative (D-) had CMV-specific T cells that are exclusively of recipient origin and significantly influenced the chimerism status toward recipients. To explore the impact of seropositivity and seronegativity of CMV in recipients and donors on early chimerism, we undertook a retrospective analysis of patients with myeloid disorders who received four days of fludarabine and busulfan with or without anti-thymocyte globulin (ATG) 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: 42 patients were identified and included in the study. All patients received fludarabine (40 mg/m2/day x 4 doses), busulfan (3.2mg/kg/dose IV x 4 doses). Of these 42 patients, 25 had anti-thymocyte globulin. There were 28 male and 14 female patients with a median age of 62 years (range 48-74yrs). Median time to follow up was 8 months (0.8-54 months). Disease risk was considered advanced in 21 patients, intermediate in 4 and early in 17. Median blast number at time of SCT was 5%. Stem cell source included peripheral blood in all patients. There were no primary graft failures. Total recipient cell chimerism showed increase or persistence of recipient chimerism in 5/11 (45%) of R+D- vs 2/6 (33.3%) of R-D- in the group of patients who received ATG, p=1.0, with a mean of recipient chimerism at day 100 of 20.4% in the R+D- group compared to a mean of 17% in the R-D- group. In the group who did not get ATG, recipient chimerism persistence or increase was not that different between the R+D- patients 3/4 (75%) compared to 4/4 (100%) in patients who were seronegative for CMV (R-), p=1.0. The mean of recipient chimerism at day 100 in the R+D- no ATG group was 23.25% with a median of 12% while the mean and median at day 100 in the R- no ATG group were 35.25% and 19.5% respectively (p=0.573).When looking at the persistence or increase in recipient chimerism in the group of patients who were R+D-, in those who got ATG it was 45% increase vs 75% increase in those who did not get ATG (p=0.569) with a median of 12% vs 0% respectively (p=0.49). Also increase or persistence of recipient chimerism was 33.3 % in patients who were R- and got ATG vs 100% in R- no ATG patients (p=0.076)with median at day 100 of 0 vs 19.5% (p=0.098).Conclusion: In this small cohort from a single center, we found that in patients with myeloid disorders who received reduced toxicity ablative conditioning regimen, the group of patients who received ATG, there was no statistically significant increase in recipient chimersim in the R+D- group compared to R-D- group. This is different from what Sellar et al found in a small group of patients who received alemtuzumab. These results may indicate a difference between ATG and alemtuzumab in the effect of CMV seropositivity and negativity on the recipient chimerism, which need to be studied further in a larger retrospective or prospective study. This is especially important in myeloid disorders since persistent or increase in recipient chimerism may identify high-risk patient cohorts who may benefit from additional therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Low-Dose Antithymocyte Globulin, Post-Transplant Cyclophosphamide and Sirolimus As Graft-Versus-Host Disease Prophylaxis in Unrelated Donor Transplants

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cure for several patients with high-risk hematological malignancies. Recent advances in supportive therapies have significantly reduced the treatment-related mortality over the last decades. Nevertheless graft-versus-host disease (GvHD) still represents a major complication, and research is needed to achieve further progress in this area of transplantation. Moreover, although initial studies suggested relative equivalence, recent and large prospective studies showed higher rates of acute and chronic GvHD, and worse survival in allo-HSCT from unrelated donors (URD) compared with HLA-identical sibling donors. Following our encouraging results in haplodentical setting with the more recently post-transplantation cyclophosphamide (PTCy) and sirolimus as GvHD prophylaxis (Cieri et al, 2015), in this study we investigated whether the incorporation of low dose anti-thymocyte globulin (ATG) to PTCy and sirolimus GvHD prophylactic approach, may alleviate some of the increased alloreactivity associated with URD transplants.We retrospectively evaluated a cohort of 21 haematological patients receiving URD allo-HSCT in the San Raffaele Haematology and BMT Unit, from 2013 to 2015.All subjects had high-risk hematologic malignancies, with the most common diagnosis being myeloid diseases (52%). A total of 11 patients had active disease at HSCT, while 9 were in hematologic remission. Nine patients scored high according to disease risk index defined by Armand et al, 10 scored intermediate, while only 2 had a low disease score.Myeloablative conditioning consisted of treosulfan (14 g/m2/day) on days -6 to -4, fludarabine (30 mg/m2/day) on days -6 to -2, and melphalan (70 mg/m2/day) on days -2 and -1, followed by T-replete G-CSF-mobilized PBSCs (n=19) or BM grafts (n=2). Whether 12 patients received stem cells from HLA- matched URD (fully 10/10 HLA matched), the remaining 9 had one-locus HLA-mismatched from their donors. During conditioning patients received ATG-Fresenius 5 mg/kg per day IV on day -4 to -2. In vivo B-cell depletion was obtained by rituximab (i.v. 375mg/m2 given as a single dose on day -1). Postgrafting immunosuppression consisted of PTCy (50 mg/kg/day) on days 3 and 4. Sirolimus was given orally from day 5, and monitored to maintain a target therapeutic plasma level of 5-14 ng/ml. According to chimeric status and evidence of GVHD, the dosage of sirolimus was tapered during the second month post-transplantation, ending in complete withdrawal within the sixth month after HSCT. Mycophenolate mofetil (MMF) was initiated orally at 10 mg/kg t.i.d. on day 5 after HSCT, and withdrawn on day 30.Median follow up was 247 days (range 29-486) from HSCT. All patients were engrafted in a median time of 17 days (range, 14-51 days), with full donor chimerism achieved by day 30. Poor graft function was observed in 11 patients. Post-HSCT recovery of lymphocyte subsets was lengthy as compared to other unmanipulated HSCT, with a median time to CD3>100/ml of 41 days (range, 22-389 days) by flow-cytometry, while 5 patients never reached immune reconstitution.All except one developed febrile neutropenia during aplasia. We observed a high rate of serious infections: severe pneumonia in 7 cases (5 required NIV, 4 were admitted to ICU), CMV reactivation in 13 (3 CMV disease), HHV6 reactivation in 10, BK virus haemorrhagic cystitis in 2 patients. Although the overall mortality associated with these complications was limited, as evidenced by a favourable NRM, it caused significant morbidity in patients and often required aggressive therapies, potentially contributing to poor graft function.The incidences of acute and chronic GVHD were low. A grade II-IV acute GvHD was reported in 10% patients, while five patients developed chronic GvHD (mild in 4 cases, only one moderate).Non-relapse mortality (NRM) at 100 days and 1 year was 4.8% and 20.7%, respectively. The estimated 1-year overall survival (OS) and relapse incidence were 51% and 22.5% respectively.Although limited by a small number of patients with relatively short follow-up, our study suggests that URD allo-HSCT with ATG, PTCy and sirolimus-based GvHD prophylaxis, can be very effective in preventing GvHD, but at the expense of delayed immune reconstitution and high rate of infectious complications, while warranting acceptable NRM and relapse incidence. DisclosuresBonini:MolMed S.p.A: Consultancy.

Donor Chimerism of B Cells and Nature Killer Cells Provides Useful Information to Predict Hematologic Relapse following Allogeneic Hematopoietic Stem Cell Transplantation.

In this study we investigated the correlation between donor chimerism status and disease relapse following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The chimerism of Fluorescence-activated cell sorter (FACS) sorted CD3+T lymphocytes of 153 cases, CD56+CD16+NK lymphocytes of 153 cases and CD19+B lymphocytes of 31 cases with acute B lymphoblastic leukemia (B-ALL) was analyzed post-transplant utilizing polymerase chain reaction amplification of short tandem repeats (PCR-STR). A total of 33 patients (33/153, 21.6%) had recurrent disease. The positive predictive values of declining donor chimerism for hematologic and isolated extramedullary relapse were 58.8% and 10% (P=0.018, Chi-Square). The positive predictive values of declining donor chimerism in BMB, BMT, BMNK and PBB for hematologic relapse were 11.6%, 0%, 0% and 0% under close monitoring in patients with B-ALL. Only the donor chimerism in BMB significantly decreased in the group with hematologic relapse as compared with the group without hematologic relapse (P=0.00, Independent-samples T test) in patients with B-ALL. The median drop of donor chimerism in PBT, BMT, PBNK and BMNK were 0%, 0%, 5.9% and 2.8% one or two weeks prior to hematologic relapse in patients with non-B-ALL. The donor chimerism in PBNK significantly decreased prior to hematologic relapse in the group with hematologic relapse as compared with the group without hematologic relapse (P=0.022, Independent-samples T test).These data suggest donor chimerism of BMB can be used to predict the occurrence of hematologic relapse in patients with B-ALL. Donor chimerism decrease in PBNKwas associated with a somewhat increased risk of hematologic relapse in patients with non-B-ALL. Therefore, our results reveal a more effective path to individually predict for hematologic relapse by dynamic monitoring different cell lineages in different disease.