Single-unit Cord Blood Transplantation Research Articles

Tacrolimus and Short-Term Methotrexate (mini-MTX) for Graft Versus Host Disease (GVHD) Prophylaxis after Unrelated Single Unit Cord Blood Transplant (CBT) in Pediatric Patients.

CBT is increasingly used in the management of patients with a variety of hematologic and neoplastic conditions. We investigated the use of Tacrolimus with MTX for the prevention of GVHD. Sixty consecutive pediatric patients (37 males, median age 6.8 years, range 0.4–20 y) underwent 63 single unit CBT. Sixty-two units were mismatched with the patients (1 Antigen mismatch [Ag MM]: 20; 2 Ag MM: 39; 3 Ag MM: 3). Five patients had non-malignant conditions (severe aplastic anemia [SAA] 2, Wiskott-Aldrich 2, Hunter 1). Thirty-one of the 55 patients with malignant diseases (ALL 30, AML 16, CML 2, NHL 2, MDS 2, JMML 2, neuroblastoma 1) had high-risk disease. The conditioning regimen was chemotherapy-only in 34 (Thiotepa, Busulfan[Bu], Cyclophosphamide[Cy] for 28 children younger than 5 years or with prior irradiation; Bu-Cy in the patient with Hunter and a patient with AML and trisomy 21; Fludarabine[Flu], Cy, Lymphoglobulin in one patient with SAA; Bu-Flu:3) transplants. Radiochemotherapy was implemented in 29 patients (Flu, Melphalan[Mel], TBI: 26; Etoposide, Mel, TBI:1; Etoposide, Mel, TBI:1; Flu, Cy, ATG, 200cGy TBI:1 SAA patient). The median pre-cryopreservation total nucleated cell dose was 4.3 x 107 / kg recipient weight (range 1.8–12 x 107/ kg). The combination of Tacrolimus and mini-MTX (5 mg/m2 on days 1, 3 and 6) was used in 60 transplants (Tacrolimus alone in 3). ATG or steroids were not routinely used.Results: Forty-six of the 52 transplants evaluable for acute GVHD did not have severe GVHD (Grade 0:11 patients; I: 10 patients; II: 25 with 22 limited to skin only; III: 3; IV: 3 patients). Eight patients had primary graft failure (2 with persistent disease). There were 3 early deaths. Chronic GVHD was diagnosed in 8/42 evaluable patients. With a median follow-up of 1,757 days (range 512–3,731) 27 patients are alive without disease.Conclusion: Tacrolimus with mini-MTX should be considered for the prevention of GVHD after mismatched unrelated cord blood transplants in pediatric patients with malignant or non-malignant diseases.

Outcomes in the Recent Seattle Cord Blood Experience: Low TRM and Relapse; High Mild Acute GVHD and CMV Reactivation.

Studies of simultaneous transplantation of two cord blood units have suggested that as compared to single unit cord blood transplantation (CBT), increased cell dose may overcome graft rejection and improve survival, particularly in mismatched CBT, as well as enhance the graft-versus-malignancy effect. Successful double CBT has been reported following both myeloablative and reduced intensity (RIT) conditioning. However, several concerns remain, including the possibility of increased incidence of GVHD compared to single unit CBT, as well as persistent delays in hematopoietic recovery and immune reconstitution despite the increase in cell dose achieved by double CBT. We have used an algorithm to guide selection of units in our most recent cohort of CBT patients (pts), such that single unit CBT is allowed only for units matched for 5/6 HLA with total nucleated cell (TNC) dose of at least 4.0 ×107/kg or matched for 6/6 HLA with TNC at least 3.0 ×107/kg. Between February 2006 and June 2007, 22 pts underwent CBT for high risk hematologic malignancies (21 in complete morphologic remission, 8 with minimal residual disease, 1 with active mycosis fungoides). Fourteen pts median age 20.5 years (range 0.6–42) underwent myeloablative transplantation following conditioning with fludarabine (FLU) 75mg/m2, cyclophosphamide (CY) 120mg/kg, and total body irradiation (TBI) 13.2 GY. Eight pts median age 52.5 years (range 24–68) underwent RIT following FLU 200mg/m2, CY 50mg/kg, and TBI 2 GY with the addition of ATG 90mg/kg if there was no exposure to chemotherapy in the three months preceding transplant. GVHD prophylaxis was cyclosporine and MMF. Donor-recipient matching based on intermediate resolution for HLA-A and B and high resolution for HLA-DRB1 was as follows: single 5/6 unit for three pts (14%), two units 4/6 for 12 pts (55%), two units 5/6 for three pts (14%), one 4/6 and one 5/6 unit for three pts (14%), and two units 6/6 for one patient (5%). All double CBT units were at least 4/6 matched to each other. At a median follow-up of 243 days among surviving pts (range 71–451), overall survival (OS) is 77% (17/22). OS is 93% (13/14) among pts treated with myeloablative conditioning and 50% (4/8) among RIT pts. 14% (3/22) of pts have relapsed. Day 100 transplant related mortality (TRM) is 5% (1/22). Incidence of grade II–IV and III–IV aGVHD in 19 of 22 pts who had sustained engraftment and survived past day 30 are 89% (17/19) and 26% (5/19) respectively. Three pts are non-evaluable due to septic death prior to engraftment, early relapse, and graft failure. 100% (11/11) of pts seropositive for CMV prior to transplant developed CMV reactivation measured by PCR at a median of 22 days (range 3–39). No (0/11) seronegative pts developed CMV. There was one case of non-fatal CMV pneumonitis. Using this strategy, we have observed limited early TRM and a lower than expected incidence of relapse in a high-risk population. We have seen a higher incidence of a GVHD than reported at other centers, and long term monitoring for chronic GVHD will be important. Though we have observed few significant infectious complications, all seropositive patients reactivated CMV early post-transplant, emphasizing the need for close monitoring of CMV reactivation and improved treatment strategies. Ongoing experience will confirm the efficacy of our algorithm for the use of two CB units for all 4/6 HLA matched units and low dose 5/6 and 6/6 units.

Use of Third Party Ancillary Cells for Enhancement of Full Donor Chimerism and Immunomodulation in Cord Blood Transplants.

We have pioneered co-infusion of a low number of T-cell highly depleted mobilized hematopoietic stem cells (MHSC) from a third party donor (TPD) as a tool to increase rates of cord blood transplant (CBT) engraftment and full chimerism in adults with high risk hematologic malignancies (“dual transplant”, Haematologica 2006; 9:640–8). The conditioning regimes used have been myeloablative although of reduced extra-hematological toxicity. After achieving very favourable results regarding both engraftment and full chimerism, we have started using this approach to evaluate the addition of other TPD cells to the purpose of optimizing CBT immune reconstitution. Results on CBT engraftment, chimerism and survival are available for analysis at this time on 53 patients (M/F 33/20, median age 35 years, range 16–60) who received units with a total cell count (TCC) of 1.1 to 4.3 x 107/Kg (median 2.3) and 0–3/6 HLA disparities, who have received TPD MHSC: 38 from an haploidentical donor, 15 from a related or unrelated donor not sharing an HLA haplotype. Days to ANC>500/uL ranged 9–36 (P50: 11; P90: 20). Initially most of the ANC was predominantly from the TPD with increasing proportions of granulocytes of CB source. Days to full CB chimerisms ranged from 11 to 97 (P50: 37; P90: 93). With a median follow up of 15 months, 3 year OS and DFS of these 53 patients are 60% and 53%. OS for patients older and younger than 40 years are 50% and 64% respectively (p= 0,37). Five patients relapsed, 2 of them achieved new complete remission maintaining full CB chimerism. Acute GVHD occurred in 19 patients, most of them grade I-II with favorable response to treatment. Four cases were grade III-IV, causing death to 3 patients. Other were toxic (VOD 2, MOF 3 and cerebral hemorrhage 1) or infections (all but one CMV). These have been the main cause of morbidity after post-transplant neutropenia and were favoured by a slow recovery of the protective immunity. Reconstitution of lymphocyte subpopulations (detailed data available for 31) is similar to what has been described for single unit CBT: prompt recovery of NK cells (1 month), followed by recovery of B cells (3 months) and slower recovery of T cells subpopulations: T8 in about one year, T4 and T-regs within the second year. No adverse effects due to the co-infusion of the TPD MHSC have been observed. Ex-vivo expanded MSC from the same TPD have been co-infused to 8 patients in addition to the MHSC. The number of co-infused MSC has ranged 1.16–3.24 x 106 cells/kg (median 1.38) without adverse effects observed so far: ANC has occurred as in the other patients and only one had signs of aGVHD, who did nor achieve stable response to antivirals for CMV, achieving continued control of both after the infusion of a new dose of 1.12 x106 cells/kg.Conclusion: These results consolidate our previous description of the “dual transplant” strategy as an approach that may allow high rates of engraftment, full chimerisms and survival of HLA mismatch CBT of relatively low cell content for adults of a wide age range with haematological malignancies, with the possibility of adding other subpopulations of the same TPD as cell therapy tools.

Ex vivo Expansion of Umbilical Cord Blood Stem Cells Using Different Combinations of Cytokines and Stromal Cells

Umbilical cord blood is a promising source of hematopoietic stem cells (HSC) for allogeneic transplantation. However, graft rejection and delayed engraftment remain major limitations, both of which are related to a limited number of stem cells in the cord blood graft. Ex vivo expansion of HSC has been suggested as one of the ways of overcoming the challenges caused by a limited hematopoietic cell number from cord blood stem cell transplantation. In this study, we quantified and characterized an ex vivo expansion capacity of cord blood-derived HSC in a liquid culture system under different conditions. These conditions included: the combinations and concentrations of hematopoietic growth factors [stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and erythropoietin (EPO)], placental conditioning medium (PCM), and stromal cell support. During culture, the mean nucleated cell count, the mean CD34+ cell count, fold expansion, viability, clonogenic assays and immunophenotypic characterization were performed on day 0, day 7, day 12 and day 14 on the expanded cellular product. The maximum expansion was achieved using GF2 (SCF + IL-3 + GM-CSF) with stromal cell support. The mean CD34+ cell expansion on days 7 and 12 was 16.25- and 21.4-fold (5.2–32), respectively, and the mean nucleated cell expansion was 15.1- and 21-fold (18.1–23.2). The mean nucleated cell viability on day 12 was 87.9% (85.6–92.5). After 12 days, granulocyte-macrophage colony-forming units CFU-GEMM showed a 20.4-fold increase. A 21.4-fold increase in the CD34+ cells and a 20-fold increase in the CFU-GEMM should provide enough cells from a single cord blood unit to reduce the period of cytopenia after single unit cord blood transplantation. Even if there was some doubt about the long-term repopulating capacity of the expanded cells part of the collected umbilical cord cells (25%) could be expanded till day 12 after transplanting the major part (75%) of the collection.

Single Unit Cord Blood Transplant Supported by Third Party Highly Purified Mobilized Hematopoietic Stem Cells: Immune Reconstitution Studies.

Background and Objectives: Following cord blood transplants (CBT) there is a period of severe and often prolonged immune deficiency that results in long term susceptibility to infections. Immune reconstitution is an important factor for long term survival. We analyzed the immune reconstitution of adult recipients of a single unit CBTs supported by a low number of third party donor highly purified mobilized hematopoietic stem cells (dual CB/TPD transplants), as previously described (Magro et al. Haematologica 2006; 91:640–8). This strategy results in transient double chimerism of CB and TPD cells and early granulocyte recovery, initially of TPD predominance. Complete CB chimerism is regularly achieved within 100 days.The objective of this study is to evaluate immune reconstitution in this CBT.Patients and Methods: Data were obtained from 19 patients between July 2004 and July 2006. Data collection was initiated at different intervals (from day −7 to +720, quartiles Q1=35, Q2=90 and Q3=210). Samples were obtained on days +15, +35, +55, +90 and monthly thereafter up to two years. By four-color flow cytometric immunophenotyping we analyzed the subsets of peripheral blood lymphocytes: CD3+/CD4+ (T helper/inducer), CD3+/CD8+ (T suppressor/cytotoxic), NK cells (CD3−/CD56+/CD16+) and B cells, as well as cells with naïve, memory and effector T-cell immunophenotypes. TREC bearing cells were analyzed by quantitative PCR in sorted CD4+ and CD8+ T cells collected from 3 months post-transplant onwards.Results: CD56+ cells recovered early after transplantation, with median absolute number counts (ANC) of 69 (range 18–307), 170 (0–366) and 159 (32–531) cells/uL in days +15, +35 and +55 samples [normal controls 153 (71–438)], representing the largest subset within the first two months (decreasing proportions of 60%, 50% and 40%, respectively). ANC of CD4 and CD8 T cells remained low for several months, progressively increasing to reach normal ranges at different intervals. Naïve CD4 and CD8 cells (CD45RO−/CD27+) start to be detected by immunophenotyping after three months post-transplantation with median ANC of 17 (12–79) and 12 (5–103) cells/uL respectively and increasing thereafter [normal controls 860 (552–1072) and 331 (227–521)]. By the end of the first year values of T cell subsets were: CD4+, 823 (16–1123) cells/uL [normal controls 872 (470–1093)]; CD8 934 (56–1174) [normal controls 371 (208–808)], with persisting predominance of the naive phenotypes and proportions of memory phenotypes slowly increasing. B cells became detectable around day +90 with median ANC of 249 (0–1934) cells/uL, rapidly reaching values within the normal range [275 (133–684)]. Transient acute GVHD was developed by six of the 19 patients. All showed a transient drop in absolute numbers of NK, T and B cells. Chimerism analysis showed initial transient double chimerism of CB and TPD cells. Complete CB chimerism was achieved between days +15 and +94 (median, +35). Results of chimerism of lymphocyte subsets and TREC are not yet available.Conclusions: Following dual CB/TPD transplants we have observed early recovery of NK and B-cells and slow development of T cells subsets and of non-naive immunophenotypes.

Post-engraftment infections in adult patients transplanted with single cord blood units supported by co-infusion of mobilized purified hematopoietic stem cells from a third party donor

Abstract We have co-infused mobilized purified hematopoietic stem cells (HSC) from a third party donor to shorten neutropenia in single unit cord blood transplantation (CBT). We describe post-engraftment infectious complications in 33 consecutive adults with high-risk hematologic malignancies. Median age was 30 (range 16–59), 22 were male. Patients were conditioned with TBI or busulfan, fludarabine, cyclophosphamide and ATG. GVHD prophylaxis included CyA and steroids. Non-bacterial prophylaxis included fluconazol, acyclovir, trimethoprim-sulfamethoxazole and azithromycin or Fansidar (toxopositive cases). Median total infused CB cell dose was 2.26 x 107/kg (1.31–3.7). Pre-CBT toxoplasma serology was positive in 12, negative in 13 (4 received HSC from a seropositive donor) and unknown in 8 cases. Pre-CBT CMV serology was positive in 30 cases. Pre-CBT, 7 patients were HBsAb(+), HBsAg(−) and 1 HBsAg(+) HBsAb(−). ANC>500/uL was achieved by 32/33, median time 10 days (9–36). Full CB chimerism was achieved in 32/33 cases. Most clinically significant infections occurred after ANC recovery (no major neutropenic infections).There were 34 episodes of CMV reactivation (3 patients developed CMV peumonitis and died Short and long term results in adult CBT are improving. However we and others are reporting high incidence of post-engraftment unusual infections which seem related to delayed recovery of cell-mediated immunity. CMV, for which early diagnostic techniques and antiviral agents are available, heads the list. Our toxoplasma cases have prompted us to use prophylaxis in seropositive patients. HBV reactivation among HBsAb(+) HBsAg(−) patients raises the issue of prophylaxis during CBT versus close serological monitoring and pre-emptive therapy. Leishmaniasis is endemic among our city's dogs, so we actively search for it in FUO (BM examination and culture). Polyomavirus can be found in the urine of up to 50% of BM recipients, viruria preceding symptoms. The value of prospective monitoring and the role of antivirals are unknown. Other unusual infections (as tripanosomiasis in our country) require high index of awareness and consideration in FUO protocols (BM, PB smears). Adult CB recipients are at very high risk of fastidious non-bacterial infections requiring wide pre-BMT screening, close analytical and clinical monitoring, prophylactic/preemptive strategies or early aggressive therapy and innovative immunotherapeutic approaches.

Post-Engraftment Infections in Adult Patients Transplanted with Single Cord Blood Units Supported by Co-Infusion of Mobilized Purified Hematopoietic Stem Cells from a Third Party Donor.

We have co-infused mobilized purified hematopoietic stem cells (HSC) from a third party donor to shorten neutropenia in single unit cord blood transplantation (CBT). We describe post-engraftment infectious complications in 33 consecutive adults with high-risk hematologic malignancies. Median age was 30 (range 16–59), 22 were male. Patients were conditioned with TBI or busulfan, fludarabine, cyclophosphamide and ATG. GVHD prophylaxis included CyA and steroids. Non-bacterial prophylaxis included fluconazol, acyclovir, trimethoprim-sulfamethoxazole and azithromycin or Fansidar (toxopositive cases). Median total infused CB cell dose was 2.26 x 107/kg (1.31–3.7). Pre-CBT toxoplasma serology was positive in 12, negative in 13 (4 received HSC from a seropositive donor) and unknown in 8 cases. Pre-CBT CMV serology was positive in 30 cases. Pre-CBT, 7 patients were HBsAb(+), HBsAg(−) and 1 HBsAg(+) HBsAb(−). ANC>500/uL was achieved by 32/33, median time 10 days (9–36). Full CB chimerism was achieved in 32/33 cases. Most clinically significant infections occurred after ANC recovery (no major neutropenic infections).There were 34 episodes of CMV reactivation (3 patients developed CMV peumonitis and died <+60).Three patients developed CNS toxoplasmosis (2 seropositive pre-CBT): 1 died as a result (day +70); 1 died of acute GVHD (+120); 1 is alive on Fansidar (+547). One patient had visceral leishmaniasis (day +180) (BM aspirate for FUO; alive on amphotericin, day +379). Seven patients developed hemorrhagic cystitis, 6 related to polyomavirus (PCR on urine): 4 resolved without specific treatment and 2 with treatment (cidofovir, leflunomide). One had asymptomatic viruria. Reappearance of HBsAg occurred in 3/7 of the HBsAb (+) HBsAg(–) patients after day 100: 1 asymptomatic, 1 acute hepatitis and 1 cirrhosis. A HBsAg(+) patient was transplanted on adefovir (no increase in the viral load or liver enzymes). Four late (≥120) episodes of HZV were seen. A 27-year-old native Spaniard died (day +39) of tripanosomiasis (BM aspirate for FUO, positive PCR). Activation of pre-CBT transfusion-related infection is suspected.Short and long term results in adult CBT are improving. However we and others are reporting high incidence of post-engraftment unusual infections which seem related to delayed recovery of cell-mediated immunity. CMV, for which early diagnostic techniques and antiviral agents are available, heads the list. Our toxoplasma cases have prompted us to use prophylaxis in seropositive patients. HBV reactivation among HBsAb(+) HBsAg(−) patients raises the issue of prophylaxis during CBT versus close serological monitoring and pre-emptive therapy. Leishmaniasis is endemic among our city's dogs, so we actively search for it in FUO (BM examination and culture). Polyomavirus can be found in the urine of up to 50% of BM recipients, viruria preceding symptoms. The value of prospective monitoring and the role of antivirals are unknown. Other unusual infections (as tripanosomiasis in our country) require high index of awareness and consideration in FUO protocols (BM, PB smears). Adult CB recipients are at very high risk of fastidious non-bacterial infections requiring wide pre-BMT screening, close analytical and clinical monitoring, prophylactic/preemptive strategies or early aggressive therapy and innovative immunotherapeutic approaches.