Early Multilineage Chimerism Predicts The ‘Winning’ Unit In Double Cord Blood Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 300-300
Author(s):  
Rachael E Hough ◽  
Bronwen E. Shaw ◽  
Pip Patrick ◽  
Nigel Russell ◽  
Antonio Pagliuca ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
B Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cord Blood Transplantation ◽  
Conditioning Regimen ◽  
British Society ◽  
Post Transplant ◽  
Kinetics Of

Abstract Background Umbilical cord blood (UCB) is an established alternative haemopoietic cell source for use in clinical transplantation for life-threatening malignant and non-malignant disorders. One challenge in using UCB in adolescents and adults has been delayed engraftment due to the finite and limited cell dose of a single unit, which has been shown to be a key determinant of engraftment, transplant related mortality and survival. The use of double unit transplantation, pioneered at the University of Minnesota, has become one of the most successful approaches to overcome this limitation to date. This group have also developed a reduced intensity conditioning (RIC) regimen, successfully broadening access to UCB transplant to older patients and those with co-morbidities. The kinetics of granulocyte, T cell and B cell chimerism in this setting require detailed study. Methods Since 2009, the British Society of Blood and Marrow Transplantation have conducted a prospective, phase II study of UCB transplantation using the Minnesota RIC conditioning regimen (Fludarabine 200mg/m2, Cyclophosphamide 50mg/kg and TBI 2Gy), with Ciclosporin and Mycophenolate Mofetil graft versus host disease prophylaxis. Lineage specific chimerism was performed at days 7, 14, 21, 28, 35, 60, 100, 180, 360 and 720 post transplant and analysed at laboratories associated with participating centres. Results 28 consecutive adult trial patients who have received a double unit transplant, have engrafted and have chimerism data up to at least day 35 are included in this analysis. The ‘winning' unit had a median unit:recipient match of 4/6 (range 4-6/6), with a median pre freeze total nucleated cell (TNC) count of 189x107 (range 83-250) and CD34 of 84x105 (range 23-169). The ‘losing' unit had a median unit:recipient match of 5/6 (range 4-6/6), with a median pre freeze total nucleated cell (TNC) count of 183x107 (range 127-303) and CD34 of 55x105 (range 42-95). Despite the low white count early post transplant, peripheral blood (PB) lineage specific chimerism for mononuclear cells (PBMC), T cells and granulocytes was feasible in nearly all patients. B cell chimerism was unsuccessful or not available in 55% of time points. The pattern of early T cell and granulocyte chimerism is summarised in the table. From day 60 onwards, the median granulocyte and T cell chimerism remained 100% winning unit. However, T cell chimerism at day 14 identified the winning unit in all patients with a result at this time point (n=25). Contribution to the B cell compartment was 100% winning unit by day 35, with 83% recipient at day 7 and 24% at day 14 and 4% losing unit at day 7 and 14. Conclusions Lineage specific chimerism is technically feasible in the immediate post transplant period and gives important insights into the kinetics of double cord blood unit engraftment. Although the ‘losing' unit may contribute to the B and T cell compartments in the first 2 weeks, it contributes little after day 21. The winning unit is clearly identifiable by day 14 in all lineages. Early granulocyte recovery (driven by G-CSF) in the RIC setting is seen to be primarily due to autologous recovery until around day 21 after which the winning unit predominates. These data provide an insight into the biology of engraftment that may also inform additional novel strategies such as ex vivo CD34 expansion and adding in haploidentical stem cells. Disclosures: No relevant conflicts of interest to declare.

Telomere Length Plays No Role in Clinical Unit Dominance After Double-Unit Cord Blood Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4661-4661
Author(s):  
Beth Ashbridge ◽  
Ahmet Zehir ◽  
Lingbo Shen ◽  
Juliet N Barker ◽  
Malcolm A.S. AS Moore
Keyword(s):  
Cord Blood ◽  
Telomere Length ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cord Blood Transplantation ◽  
Length Variation ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Percentage Loss

Abstract Abstract 4661 Background: Umbilical cord blood (CB) is an alternative source of allogeneic hematopoietic stem cells for transplantation and has the advantage of a reduced stringency of the required human leukocyte antigen-match. It is limited, however, by low total nucleated cell (TNC) dose and a low progenitor number in single CB units, thus restricting the use of CB transplantation (CBT) in larger children and adults. One strategy to augment engraftment is to combine 2 units from 2 different donors in a double-unit graft. We have previously shown that in most cases, one unit emerges as the sole source of hematopoiesis long-term, but as yet the mechanism of unit dominance remains unknown (Blood, 2010, 116(19):3999–4006). CB units are known to have an inherent biological variation in telomere length, the repeat sequence capping the ends of chromosomes. Telomere length variation and progressive shortening of telomere could later hematopoietic potential. Methods: We evaluated if telomere length has a role in unit dominance and how telomere length progresses over time post-transplant. We purified mononuclear cells from small aliquots of each unit of double-unit grafts and post-transplant peripheral blood (days +28, 100, 180 and 1 year) in 12 adult double-unit CBT recipients transplanted for hematologic malignancies at MSKCC. Average telomere length was measured using the TeloTTAGGG Telomere Length Assay (Roche) which utilizes Southern analysis of terminal restriction fragments (TRF) that are obtained by the digestion of isolated genomic DNA. Results: All 12 patients engrafted with one unit showing predominance. While there was a range of telomere length (5.57–12.09 kb) in the 24 units evaluated on the day of transplant, when comparing the telomere length of engrafting to non-engrafting units, there was no association between telomere length at day 0 and subsequent unit dominance. In 5 patients the engrafting unit had longer telomeres and in 7 patients the non-engrafting unit had longer telomeres. In serial assays, 7 of 12 patients demonstrated telomere length stabilization post-transplant in the dominant unit with a mean percentage loss of telomere length of 7.15% ± 2.33%. A second group (n = 5) demonstrated a decrease in telomere length with a mean percentage loss of 29.48% ± 5.12%. While this difference is significant (p = 0.0015) the clinical significance of this finding is uncertain. We are currently following these patients in order to correlate telomere length (stabilization versus decrease) with clinical outcomes. Conclusions: This data suggests that unit dominance is not influenced by telomere length. It is likely, based on emerging data from our laboratory as well as others, that unit dominance is immune mediated. However, the influence of telomere length on the quality of engraftment is of interest and analysis of this is ongoing with larger numbers of patients required to also consider the infused cell dose. It is notable that 7/12 patients had telomere length stabilization which correlates with the high levels of telomerase activity previously reported in the in vitro expansion of CB (Blood, 2004, 103(12):4440-8). Disclosures: No relevant conflicts of interest to declare.

Vaccine Response In Recipients of HLA Mismatched Unrelated Double Unit Cord Blood Transplantation (CBT).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1247-1247 ◽  
Author(s):  
Trudy N Small ◽  
Christine Scura Iovino ◽  
Michelle Abboud ◽  
Marissa Lubin ◽  
Esperanza Papadopoulos ◽  
...  
Keyword(s):  
Hepatitis B ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cord Blood Transplantation ◽  
Chronic Gvhd ◽  
Optimal Schedule ◽  
High Dose ◽  
Vaccine Response ◽  
Post Transplant ◽  
History Of

Abstract Abstract 1247 The use of CBT has increased steadily over the last decade, with recent studies showing long-term progression-free survival similar to that of unrelated volunteer donor transplant recipients. The ability of CBT survivors to respond to post-transplant immunizations may differ from other allogeneic transplant populations due to the lack of transfer of memory T- and B-cells with the graft. Limited data have been reported on vaccine responses following this treatment modality. We, therefore, analyzed responses to immunizations in 23 double-unit CB recipients (17 adults, 6 children) transplanted at our center from 10/05-12/08. Patients were transplanted at a median age of 34 years (range 7–61) for the treatment of acute leukemia (n=13), or lymphoma/CLL (n=8/2). They received high-dose myeloablative (n=11), reduced intensity myeloablative (n=5), or non-myeloablative (n=7) conditioning according to diagnosis, age, prior therapy, and co-morbidities. GVHD prophylaxis consisted of a calcineurin inhibitor and mycophenolate mofetil. No patient received ATG. The study patients had sustained engraftment with a 5/6 (n=12) or 4/6 (n=11) HLA-matched unit. Seven recipients received rituximab (median 4 doses, range: 4–8) as planned post-transplant therapy for B-cell malignancies (n=6) or treatment of an autoimmune hemolytic anemia (n=1). Eleven patients had a history of grade II-IV acute GVHD and 5 had ongoing late acute or chronic GVHD prior to vaccination. Criteria for vaccination were: CD4 cell count of at least 200 cells/ul, PHA of greater than 60% lower limit of normal and serum IgG level >500 mg/dl at least 6 weeks following the last dose of IVIG. The median time to vaccination was 1.26 years post-CBT; this time was significantly longer in patients treated with Rituximab compared to those who were not (1.6 years versus 1.2 years, p=0.02), due to delayed normalization of B-cell numbers in the former group (449 days vs 108 days, p=0.004).Pre-vaccination titers obtained at a median of 1 year post-CBT demonstrated that over 85% of patients lacked protection against Pneumococcus, H. influenzae, and Pertussis, and at least 50% lacked immunity against tetanus, measles, and mumps. Seroconversion or >3- fold rise in titer was observed in response to tetanus, diphtheria, H. influenzae, and Pneumococcus in 90% (18/20), 81% (13/16), 80% (16/20)and 90% (18/20) of patients and was not significantly different in patients with or without a history of acute or chronic GVHD. Whereas 90% (5/6) of patients without a history of GVHD responded to a series of Hepatitis B immunizations, only 22% (2/9) of those with prior acute and/or chronic GVHD did so (p=0.03). No patient was protected against pertussis following a single TDaP (n=14) and only 1 of 5 patients responded to the protein conjugated meningococcal vaccine. Immunization with a live attenuated vaccine was initiated in 7 seronegative patients, including all 6 children, at a median of 2.25 years (range 1.5–3.6) post-CBT (MMR, n=7, Varivax, n=3). Seroconversion against measles, mumps, rubella, or chickenpox was observed in 3/7, 2/7, 6/7, and 1/3 patients, respectively. There were no serious reactions to any vaccine. These data suggest that CBT recipients are capable of responding to tetanus, diphtheria, H. Influenza and pneumococcal vaccines similar to other transplant groups. Nonetheless, the sub-optimal response to pathogens associated with outbreaks in the community (Hepatitis B, Pertussis, meningococcus, measles, mumps, varicella) highlight the need to obtain pre- and post-vaccination titers to document response, as well as define the optimal schedule of post-transplant immunizations specifically in this transplant population. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 2% of Healthy Newborns Reveal ETV6-RUNX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4082-4082
Author(s):  
Daniel Schaefer ◽  
Marianne Olsen ◽  
Ulrik Lausten-Thomsen ◽  
Cyrill Schipp ◽  
Martin Stanulla ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cord Blood ◽  
B Cell ◽  
Childhood Leukemia ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Inverse Pcr ◽  
Blood Samples ◽  
Chimeric Transcription Factor

Abstract Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns. The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected. Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened. Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented. Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. Table Table. Disclosures No relevant conflicts of interest to declare.

Reduced-Intensity Cord Blood Transplantation (RICBT) Is a Feasible Approach for Advanced Adult T-Cell Leukemia (ATL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5448-5448 ◽  
Author(s):  
Atsushi Wake ◽  
Daisuke Kato ◽  
Shinsuke Takagi ◽  
Tomomi Kawano ◽  
Eiji Kusumi ◽  
...  
Keyword(s):  
Cord Blood ◽  
Early Stage ◽  
Cord Blood Transplantation ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Late Relapse ◽  
Acute Type ◽  
Advanced Stage ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Introduction: ATL in advanced stage is a lymphoid malignancy with poor prognosis, its mean survival time being a few months. Allogeneic hematopoietic stem cell transplantation has been shown to be potentially curative approach, but the availability of HLA-matched donor, either of related or unrelated, limits its application to many of the patients. Cord blood has been widely used as an alternative donor cells. We report here the feasibility of RICBT for patients with advanced ATL. Patients and Methods: Eighteen patients with advanced ATL, including 11 acute type and 7 lymphoma type, who underwent RICBT between March 2002 and June 2005 at our institution, were retrospectively analyzed. Eighty percent of them were chemo-refractory at the time of transplant. Median age of the patients was 59 years (27–79). Pretransplant conditioning regimen consisted of fludarabine 125 mg/m2, melphalan 80 mg/m2 and TBI 4 Gy. GVHD prophylaxis was either cyclosporine (CSP, n=11) or tacrolimus (TAC, n=7) alone. The median number of infused nucleated cells and CD34 positive cells were 2.83 (1.95–4.83) x 107 and 1.00 (0.40–2.91) x 105, respectively. All the patients received CB units with HLA mismatches at 1 (n=8) or 2 (n=10) loci. Results: Neutrophil and platelet engraftment were observed in 15 (83.3%, median 16 days) and 14 patients (77.8 %, median 42 days). Two died before engraftment. Five of the engrafted patients (30%) developed acute GVHD (grade II–IV). Although 14 out of 15 patients who survived over 30 days achieved complete remission, 6 died of non-relapse mortality (NRM) within 100 days post-transplant (5 sepsis, 1 encephalitis), and another 6 died of relapse (median 225 days post-transplant). Five of the 7 patients who were alive beyond 100 days developed chronic GVHD (4 limited, 1 extensive). One patient experienced rapid tumor regression along with the chronic GVHD after cessation of TAC at day 146 post-transplant, indicating possible GvATL effect. Estimated 1-year overall and progression-free survival rates were 27.9 +/− 9.0 % and 17.2 +/− 12.8 %, respectively. Among 9 survivors beyond 100 days post-transplant, 5 remain alive at median follow-up of 17 months but only 2 of them remain progression free. Univariate analyses revealed high age (over 60), poor ECOG performance status (over 2) and high sIL-2R level (over 10000IU/L) as poor factors for survival. TAC dramatically decreased the day 100 mortality (14.3%) compared with CSP (45.5%). Conclusion: This pilot study indicates that our RICBT is feasible even for the ATL patients in advanced stage. Day 100 mortality was improved by using TAC but eventually the overall survival decreased to comparable level with CSP. To further improve the outcome, RICBT should be investigated for patients in early stage of the disease, or new approach to prevent late relapse should be explored.

In Vitro Assessment of Tolerance and Rejection Occurring After Co-Transplantation of Allogeneic Umbilical Cord Blood and Haploidentical CD34+ Cells as Treatment for Severe Aplastic Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2352-2352
Author(s):  
Nicole J. Gormley ◽  
Aleah Smith ◽  
Maria Berg ◽  
Lisa Cook ◽  
Catalina Ramos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Neutrophil Recovery ◽  
Post Transplant ◽  
Blood Transplantation ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Abstract 2352 Introduction/Methods: The administration of highly purified haploidentical peripheral blood CD34+ cells combined with an unrelated cord blood transplant results in earlier neutrophil engraftment than is typically seen with a cord blood transplant alone. Chimerism data from pilot trials evaluating this strategy have reported 3 phases of engraftment: 1) early myeloid engraftment from transplanted haplo-CD34+ cells followed by 2) cord blood engraftment resulting in dual chimerism and 3) the subsequent disappearance of haploidentical donor cells with resultant full donor cord chimerism. The mechanism accounting for the disappearance of haploidentical cells has not been defined. Here the clinical results and an in vitro assessment of alloreactivity in three patients that underwent combined haploidentical CD34+ cell and cord blood transplantation for severe aplastic anemia (SAA) are described. The conditioning regimen consisted of cyclophosphamide (60mg/kg/day on days -7 and -6), fludarabine (25mg/m2/day on days -5 to -1), horse ATG (40mg/kg/day on days -5 to -2), and total body irradiation (200cGy on day -1). GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil. PCR of STRs was used to assess chimerism in T-cell and myeloid lineages and mixed lymphocyte reaction assays(MLR) were performed on peripheral blood samples collected at different time-points post-transplant to assess for alloreactivity against the recipient, the haploidentical donor, or the cord unit. Stimulator cord blood cells for the MLR were obtained from residual cord blood cells remaining in the infusion bag after patient administration and expanded in vitro using anti-CD28/CD3 Dynabeads. Results: Prior to transplantation, all three pts had transfusion dependent SAA associated with severe neutropenia that was refractory to conventional immunosuppressive therapy. Pt 1 had an early transient myeloid recovery (ANC 400 on day+11) from the haploidentical donor followed by engraftment of the cord unit (Cord ANC > 500) on day 21. The patient is currently 2 years post transplant and has 100% cord blood chimerism and is transfusion independent. An MLR assay performed when donor T-cell chimerism was 100% cord, showed evidence for rejection of the haploid cells by cord blood T-cells, with the MLR response to haploidentical donor cells being seven fold higher than the response to fully HLA-mismatched 3rd party cells. In pt 2, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing no evidence for cord engraftment in either myeloid or T-cell lineages at any point post-transplant. The patient is currently 15 months post transplant and is transfusion independent with normal blood counts and sustained “split” chimerism (T-cells recipient in origin with myeloid cells being 100% haploidentical donor). MLR assays showed that the recipient was tolerant to the haploid donor, with no statistically significant difference in the alloreactive response to the haploid donor compared to self. In pt 3, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing split chimerism (T-cell chimerism >90% cord and myeloid chimerism 88–100% haploid donor in origin). MLR assays again showed evidence of rejection of the haploid cells by cord blood T-cells, with a trend towards greater alloreactivity against the haploid donor compared to an HLA mismatched 3rd party on post-transplant day +63. Conclusions: Combined haploidentical CD34+ cell and unrelated cord blood transplantation following highly immunosuppressive conditioning represents a viable treatment option for patients with SAA who lack an HLA-matched donor. Using this approach, 2 of 3 pts had cord blood engraftment associated with early neutrophil recovery from the haploidentical donor. In one pt, the cord unit failed to engraft. Remarkably, sustained engraftment from the haploidentical donor in this pt resulted in transfusion independence. MLR appears to be a useful approach to assess the in vitro alloreactivity of this unique stem cell graft source. In the two pts who had cord engraftment, in vitro MLR assessments established that the disappearance of haploid cells occurred as a consequence of rejection of the haploidentical cells by engrafting cord blood T-cells, rather than from non-immunological haploidentical cell graft failure. Disclosures: No relevant conflicts of interest to declare.

Unrelated Umbilical Cord Blood Transplantation for Hematological Malignancies Using Fludarabine/BUCY2 Conditioning Regimen

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4572-4572
Author(s):  
Zimin Sun ◽  
Huilan Liu ◽  
Liangquan Geng ◽  
Xingbing Wang ◽  
Kaiyang Ding ◽  
...  
Keyword(s):  
Cord Blood ◽  
Graft Failure ◽  
Conflicts Of Interest ◽  
Cord Blood Transplantation ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Hematologic Malignancies ◽  
Median Number ◽  
Myelogenous Leukemia ◽  
Blood Transplantation

Abstract Abstract 4572 Cord blood transplantation (CBT) is largely used to treat patients affected by hematological malignant disorders. Myeloablative TBI-based conditioning appears to provide reliable engraftment after CBT for malignancies. However, the toxicity of TBI limits their widespread use. So far, a standard non-TBI based regimen has not been firmly established. In order to overcome graft failure, we investigated a strategy using Fludarabine (FLU)/BUCY2 regimen in CBT for patients with hematologic malignancies. Seventeen patients(children 16, adult 1) with hematologic malignancies who underwent single-unit CBT used a conditioning regimen comprising FLU 120 ‡r/‡u, intravenous busulfan (BU) 12.8‡r/kg and cyclophosphamide (CY)120 mg/kg (FLU/BUCY2). All patients were given a combination of cyclosporine A and mycophenolate mofetil for graft-versus-host disease (GVHD) prophylaxis. Seventeen patients with acute leukemia (n=13), chronic myelogenous leukemia (n=4) were treated, thirteen of whom were high risk diseases and two were advanced-stage at CBT. Seventeen patients with a median age of 8 years (range,2.5–46 years) and a median weight of 32 kg (range, 12–55 kg)received the median number of nucleated cells and CD34+cells infused were 5.70× 107/kg (range: 3.15–9.60×107/kg) and 3.84× 105/kg (range:1.27–5.24 ×105/kg), respectively. The cumulative incidence of primary donor engraftment was 94% (16 patients); one patient had secondary graft failure. Median time to neutrophil≥0.5×109/L was 17 days (range 12–30) and platelet engraftment (≥20×109/L) was 35 days (range 14–56). Preengraftment syndrome (PES) developed in 71% of the patients at a median of 7days (range: 5–13).9 cases developed acute GVHD (56%), more than grade II in three cases. Two of fourteen patients who survived more than 100 days developed chronic GVHD. 12 cases are alive at a median follow-up of 7 months (range 3~ 11).The probability of overall survival at 100 days and 1 year are 88.2% and 67.9%, respectively. Two cases had extramedullary relapsed. Five cases died of severe GVHD (n=3), pulmonary toxicity (n=1) and secondary graft failure (n=1). Preliminary evidence of the small study suggests successful engraftment and decreasing relapse rate following FLU/BUCY2 regimen for CBT in patients with hematologic malignancies. But it had a tendency towards increasing the incidence of GVHD-related morbidity and mortality. Whether this regimen offers a survival benefit for patients with poor-risk leukemia has to be tested in larger prospective trials. Disclosures: No relevant conflicts of interest to declare.

Differences in the Kinetics of Hematopoietic and Stem Cell (SC) Engraftment Following Murine Newborn/Cord Blood (NB) and Bone Marrow (BM) Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4949-4949
Author(s):  
Tao Du ◽  
George F. Atweh ◽  
Yelena Galperin ◽  
Rona Singer Weinberg
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Cord Blood Transplantation ◽  
Platelet Recovery ◽  
Platelet Counts ◽  
Blood Transplantation ◽  
Cell Counts ◽  
Kinetics Of

Abstract Cord blood is being used at an increasing frequency as a source of stem and progenitor cells in human hematopoietic transplantation. However, very little is known about the kinetics of engraftment of cord blood relative to bone marrow derived stem cells. We have used a murine model of newborn/cord blood transplantation to address this question. Our studies demonstrated that murine NB can provide long-term engraftment in primary, secondary, and tertiary transplant recipients. Although NB engrafts more slowly than BM, at one year, engraftment was similar in both types of recipients. We compared the rate of recovery of blood counts, hematopoietic progenitor counts and putative stem cell [SC] counts (i.e. Sca-1+, c-kit+, Lin- cells) in recipients of NB and BM transplantation. Interestingly, the SC ratio in mononuclear cells from donor NB/donor BM was 3.25 ± 0.8 (range = 2.45 – 4.75, n=15). Similarly, donor NB mononuclear cells contained approximately 30% of the number of megakaryocytic progenitors, 12% of the myeloid progenitors and 5% of the erythroid progenitors of donor BM mononuclear cells. The repopulation kinetics in recipients of donor BM and NB transplantation were analyzed at regular intervals, up to 8 months after transplantation. During the first two weeks, NB recipients had lower hemoglobin, WBC and platelet counts than BM recipients. However, by 1 month, the hemoglobin and WBC counts were at similar levels in NB and BM recipients. In contrast, NB transplantation recipients required 2 to 3 months to achieve platelet counts similar to those in BM recipients. These results are reminiscent of the well-known delayed platelet recovery following human cord blood transplantation. Progenitor cell counts in the bone marrow of recipients paralleled the hematological recovery described above. At 2 weeks post-transplantation, progenitor counts of all lineages in NB recipients were 25 to 35% of those in BM recipients. By 1 month, erythroid and myeloid progenitor numbers were similar in NB and BM recipients. In contrast, the appearance of megakaryocytic progenitors was delayed following NB transplantation and did not reach the same level as BM recipients until about 2 months after transplantation. During recovery, the number of SC in the bone marrow of both types of recipients increased gradually over time. At one month after transplantation, the number of SC in BM recipients was significantly greater than that in NB recipients, with a ratio of 4.2 ± 0.2. This SC ratio decreased gradually during the next several months. At 2, 4, and 8 months following transplantation, the ratios of SC in BM recipients/NB recipients were 3.5 ± 0.4, 2.6 ± 0.5, and 2.2 ± 0.3, respectively (n=5). This gradual decrease in the ratio of BM/NB SC suggests that NB SC increase more rapidly than BM SC. In conclusion, these data demonstrate that the kinetics of hematopoietic and stem cell recovery following NB and BM transplantation are significantly different. Although hematopoietic recovery after NB transplantation is slow at first, final engraftment is similar following NB and BM transplantation. Furthermore, the number of SC in NB recipients increases at a faster rate than the number of SC in BM recipients. These differences in SC recovery may be a reflection of differences either in the homing capacity or in the functional maturity of NB relative to BM SC. Further investigation is required to distinguish between these two possibilities.

Improving Immune Reconstitution After Cord Blood Transplantation Using Ex Vivo Expanded Virus-Specific T Cells: A Phase I Clinical Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 224-224 ◽  
Author(s):  
Patrick J Hanley ◽  
Caridad Martinez ◽  
Kathryn Leung ◽  
Barbara Savoldo ◽  
Gianpietro Dotti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Dose Level ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Cord Blood Transplantation ◽  
Viral Reactivation ◽  
Blood Transplantation

Abstract Abstract 224 Adenovirus(Ad), Cytomegalovirus(CMV) and Epstein-Barr virus(EBV) frequently cause severe morbidity and mortality in patients(pts) after stem cell transplantation (SCT) and cord blood transplantation(CBT). We have shown that adoptive immunotherapy with peripheral blood(PB) donor derived multivirus-specific Cytotoxic T Lymphocytes directed against Ad, CMV and EBV can effectively prevent and treat the clinical manifestations of these viruses after SCT. CBT, while less likely to cause GvHD than conventional SCT, is unlikely to provide passive transfer of virus-specific CTL, since CBTs come from virus-naïve donors. Here we report for the first time the transfer of CB-derived multivirus-specific CTL(cbmCTL) to CBT recipients to restore cellular immunity to Ad, CMV and EBV. The development of cbmCTLs for pts undergoing CBT requires the priming and extensive expansion of naïve T cells rather than the more limited and simple direct expansion of pre-existing memory T cell populations from virus-exposed donors. We hypothesize that cbmCTL, derived from naïve T cells, will be efficacious and persist in vivo. Our protocol uses an initial round of stimulation with autologous CB-derived dendritic cells transduced with a recombinant Ad5f35 vector containing a transgene for the immunodominant CMV antigen, pp65 (Ad5f35pp65) in the presence of IL-7, IL-12 (CTEP-NCI) and IL-15. This is followed by 2 rounds of weekly stimulation with autologous Ad5f35pp65-transduced EBV-LCL in the presence of IL-15 or IL-2. Seven cbmCTL cultures generated for clinical use contained a mean of 48% CD8+, and 36% CD4+ cells with a mean of 33% CD45RA-/CD62L+ central memory T cells. In 51Cr release and/or IFNg ELISPOT assays, cbmCTL lines showed specific activity against all viruses. We have treated 7 pts who received the 80% fraction of a fractionated CB unit followed by cbmCTLs generated from the remaining 20% fraction; two pts were treated on each dose level;5×106/m2; 1×107/m2; and 1.5×107/m2 while one pt has been treated with 2.5×107/m2 – dose level 4. Pts received cbmCTLs on days 63–146 after CBT (median: day 83). No early infusion-related toxicities or subsequent GvHD was observed. All pts engrafted neutrophils by day 30 (median: day 20) despite receiving only 80% of the CB unit. Five of 7 pts had no initial infection or reactivation episodes, remaining free of CMV, EBV, and Ad from 2 months to 2 years post-CBT. Of the two remaining pts, pt 1 was transiently viremic for CMV pre-infusion and became highly viremic 4-weeks post-cbmCTL. The pt received a 2nd dose of cbmCTLs and CMV DNA/antigen became undetectable in the PB within 16 days of the 2nd dose and remains asymptomatic and virus free >2 yr post-CBT. Analysis of this pt's PB showed a rise in CMV-T cells even prior to cbmCTL #2, with a 31-fold expansion of CMV-T cells by 4 weeks after the initial CTLs. This pt also had AdV in his stool, which resolved without additional therapy. Shortly after CTL infusion, pt 4 had detectable EBV DNA in the PB that was controlled without additional antiviral therapy. The transferred cells had long-term persistence, since T cell receptor(TCR) deep-sequencing (ImmunoSEQ) allowed us to track infused T cell clones (i.e. clones present in the infused cbmCTL but absent in peripheral blood before cbmCTL infusion) up to 1 year post-CBT in 5/5 pts tested. In summary, none of the recipients of cbmCTL developed viral disease; in two pts with viral infections, the infections resolved without progression to disease, coinciding with the appearance of virus-specific T cells in peripheral blood. Hence, administration of cbmCTL to pts after CBT has so far been safe and can facilitate reconstitution of virus-specific T cells and control viral reactivation/infection in vivo. Disclosures: No relevant conflicts of interest to declare.

HHV-6 Viremia Is Frequent but the Incidence of Encephalitis Is Low in Double-Unit Cord Blood Transplant Recipients Transplanted without ATG

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 459-459
Author(s):  
Amanda L. Olson ◽  
Parastoo B. Dahi ◽  
Junting Zheng ◽  
Sean Devlin ◽  
Marissa N. Lubin ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Limit Of Detection ◽  
Cord Blood Transplantation ◽  
Prospective Trial ◽  
Conditioning Regimen ◽  
Cox Regression Analysis ◽  
Peak Viremia ◽  
Blood Transplantation ◽  
High Level

Abstract Abstract 459 Background: While cord blood transplantation is a known risk factor for Human Herpesvirus-6 (HHV-6) reactivation, recent studies have yielded inconsistent results in regard to the level of viremia which is associated with a high risk of HHV-6 encephalitis. Moreover, the association with graft failure or transplant-related mortality is controversial. Methods: We conducted a retrospective analysis of 125 patients who underwent double-unit cord blood transplantation (DCBT) for the treatment of hematologic malignancies from 2/2006-3/2012 who were monitored for HHV-6 reactivation to examine the incidence and severity of HHV-6 viremia, the incidence of encephalitis, and the association with DCBT outcome. HHV-6 viremia was measured by quantitive PCR of HHV-6 DNA from plasma (lower limit of detection 100 DNA copies/ml). Results: Of the 125 patients (median age 42, range 1–69), 93 (74%) received myeloablative conditioning and 32 (26%) received a non-myeloablative regimen followed by 4–6/6 HLA-A, B antigen, DRB1 allele matched DCBT for the treatment of AML (N = 43, 34%), ALL (N = 24, 19%), MDS/CML/other leukemia (N = 12, 10%), or lymphoma/CLL (N = 46, 37%). No patient received anti-thymocyte globulin (ATG). Of 125 monitored patients, 117 (94%) reactivated HHV-6 to a median peak of 7,600 (range 100–160,000) copies/ml at a median onset of 20 days (range 10–59) post-DCBT. The median time to peak viremia was 23 days (range 12–62) with a median viremia duration of 10 days (range 1–60 days). Fifty-one patients (41% of total, 44% of viremic patients) developed HHV-6 > 10,000 copies/ml (median peak viremia 31,200 copies/ml at 20 days, range 12–57). Only 6 patients (5% of total, 5% of viremic patients) developed HHV-6 > 100,000 copies/ml (median peak 130,000 copies/ml at 19 days, range 14–29). HHV-6 encephalitis occurred in 2 patients (1.6%, peak viremias 13,000 and 118,000, respectively). One patient died from encephalitis and the other had a complete recovery following therapy. Four additional viremic patients had HHV-6 isolated from bronchoalveolar lavage but did not meet criteria for HHV-6 pneumonia. Using a high level viremia definition of > 10,000 copies/ml from days 14–60 (a level reportedly associated with end-organ disease and decreased survival, Dulery, BBMT 2011), viremia was not associated with diagnosis or conditioning regimen. Engrafting unit-recipient HLA-match and TNC, CD34+ and CD3+ cell dose were not associated with high level viremia. Treating viremia as a time-dependent covariate in Cox regression analysis, no association was found between viremia and neutrophil or platelet engraftment. Specifically, there were two graft failures in patients with no or low level viremia and two in patients with high level viremia. There was also no association between viremia and CMV reactivation, day 100 grade II-IV aGVHD, day 100 TRM, relapse or overall survival. A second analysis was performed to examine effects of high level viremia defined as a level > 25,000 copies/ml (N = 31 patients, the highest peak viremia quartile during days 14–60) and no associations with DCBT outcomes were detected. Conclusions: In our DCBT population, nearly all patients have reactivated HHV-6. However, the incidence of end-organ disease is relatively low. We postulate the difference between our findings and other studies reporting higher rates of HHV-6 encephalitis in DCBT recipients could be due to our exclusion of ATG from the conditioning regimen. At this time our understanding of the significance of HHV-6 after CBT is incomplete. We are currently evaluating anti-viral treatment responses, and ultimately a prospective trial is needed to better define the causality between HHV-6 viremia and transplantation outcomes, and to investigate the risk-benefits of pre-emptive therapy. Disclosures: No relevant conflicts of interest to declare.

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