Median Total Nucleated Cell Count Research Articles

Single CBT As 2nd Allo-SCT Is Promising for Selected Patients with Relapsed Hematopoietic Malignancies

Background: Hematopoietic malignancies that relapse after allogeneic stem cell transplantation (allo-SCT) generally have a poor prognosis, and there is no standard treatment. Second allo-SCT is a potentially curative treatment, but many patients do not receive 2nd allo-SCT at the best time because of the long preparation time for transplantation, and even if they do, treatment-related complications and relapse of the underlying disease often lead them to fatal outcomes. Umbilical cord blood is considered to be a promising source of 2nd allo-SCT because of its short preparation time, rapid and high graft-versus Leukemia (GVL) effect after transplantation, and better response to GVHD treatment compared to other donor sources. Therefore, we investigated the usefulness of cord blood transplantation for relapsed hematopoietic malignancies after allo-SCT. Methods: We retrospectively analyzed the outcomes of patients with hematopoietic malignancies who received single cord blood transplantation (CBT) as 2nd allo-SCT for relapse after allo-SCT in Toranomon Hospital between Apr. 1999 and Mar. 2022. We exclude the patients with Eastern Cooperative Oncology Group (ECOG) performance status(PS) of 4. Results: 263 patients were included in this study. There were 179 patients with myeloid neoplasms and 84 with lymphoid neoplasms. Median age was 45 years (range: 18-71). 239 patients were in non-remission and 24 in remission before SCT. Median interval between 1st SCT and 2nd SCT was 293 days (26-4703). Donor sources of 1st allo-SCT were related donor in 80, unrelated donor in 62, and sCBT in 121 patients. 135 patients received myeloablative conditioning for 2nd allo-SCT, and 128 patients underwent reduced intensity-conditioning. About cord blood as the donor source of 2nd allo-SCT, the median total nucleated cell count at cryopreservation was 2.71(×10 7)/kg (1.67-5.85), and median CD34 + cell count at cryopreservation was 0.97(×10 5)/kg (0.25-3.18). Median observation period of survivors was 1726 (122-6286) days after 2nd allo-SCT. Overall survival (OS), non-relapse mortality and relapse at 2-year were 16.7%, 46.9% and 40.6%, respectively. Younger patients (< 50 years) had significantly better OS than older patients (50 years and more), with 2-year OS of 21.5% and 9.9%, respectively (P < 0.001). Patients with good PS (0 and 1) had significantly better survival than those with poor PS(2 and 3), with 2-year OS of 23.4% and 2.4%, respectively (P<0.001). Patients with myeloid malignancies tended to have better survival than those with lymphoid malignancies, with 2-year survival rates of 19.2% and 11.9%, respectively (P=0.341). Patients with longer interval between SCTs (>1 year) had significantly better survival than those with shorter interval (<1 year), with 2-year OS of 23.6% and 11.6%, respectively (P=0.00443). In multivariate analysis, younger age (<50), good PS (0-1), long interval between SCTs(≧1 year) showed a superior OS. In the younger age population, patients with myeloid neoplasms showed a superior OS compared to those with lymphoid neoplasms (2yrOS: 30.3% vs 10.6%, P=0.00318). Furthermore, patients with myeloid neoplasms having good PS and long interval between SCTs showed an extremely high survival rate of 50.5% at 2 years (n=31)(Figure 1). In the elderly population, patients with long interval and good PS showed a relatively good OS of 31.4% at 2 years (n=28)(Figure 2). The type of disease (myeloid vs lymphoid) didn't have a significant influence on OS in this subpopulation (28.1% vs 50.0%, P=0.129). Conclusion: Although the outcomes of CBT as 2nd allo-SCT haven't been satisfactory, there are certain populations, including elderly patients, with favorable OS.

Minimal dose of hematopoietic stem cell transplantation without myelosuppressive conditioning for T-B+NK- severe combined immunodeficiency

We reviewed the medical records of five patients with T-B+NK- severe combined immunodeficiency (SCID) who received minimal dose allogeneic hematopoietic cell transplantation (HCT) (total nucleated cell count (TNC) lower than 1.0 × 108/kg). Patients were administered a median of 5.0 mL of bone marrow or peripheral blood without conditioning (in four) or with anti-thymocyte globulin alone (in one). Three patients received HCT from a matched sibling donor, one from unrelated donor, and one from familial mismatched donor. The median TNC and CD34+ cells were 0.54 (0.29–0.84) × 108/kg and 0.61 (0.35–0.84) × 106/kg, respectively. Engraftment was achieved in all. Total T cell, CD4+ cell, and CD8+ cell recovery was obtained within a year in four, and immunoglobulin replacement was discontinued in all. All patients survived, exhibiting stable donor chimerism. We obtained sufficient therapeutic effects with minimal dose transplantation without intensive conditioning in patients with T-B+NK- SCID.

Haploidentical Hematopoietic Stem Cell Transplantation With Reduced Intensity Conditioning, T-Cell Replete Grafts and Post-Transplant Cyclophosphamide In Pediatrics

Hematopoietic stem cell transplants (HSCT) may be curative in the treatment of many hematological diseases. However, most patients do not have an HLA- matched sibling donor and an unrelated donor search may be a long process, during which the patients' disease may relapse or progress. Haploidentical transplants with in vitro T-cell depletion have been used in Pediatrics. However, it requires specialized laboratory infra-structure to manipulate the graft, increases the chances of graft failure and relapse. In vivo T-cell depletion can be achieved with the use of post-transplant cyclophosphamide, as reported by the Johns Hopkins and other groups. The results in adults are very promising and comparable to unrelated double cord blood transplants. Children, however, have not been included in any of the reported protocols. Our objective is to report the institutional experience with five consecutive children who received Haploidentical HSCT with reduced intensity conditioning, T-cell replete grafts and post transplant cyclophosphamide. MethodsChildren with clear indication to be treated with HSCT and no HLA-matched related or unrelated donor available were offered compassionate haploidentical transplants. Conditioning comprised cyclophosphamide 14.5 mg/kg/day on days -6 and -5, fludarabine 30 mg/m2/day for five consecutive days starting on day -6, and 2Gy total body irradiation given in a single fraction on day -1. Bone marrow was harvested from donors and infused into recipients on day 0. The target nucleated cell dose was 5 x10(8) mononuclear marrow cells/kg (i.e., peripheral blood count was substracted from the marrow cell count). There was no graft manipulation. GVHD prophylaxis consisted of cyclophosphamide 50 mg/kg IV on days +3 and +4 with 140% Mesna divided on hours 0, 4, 8 and 12 after each dose, Mycophenolate mofetil 15 mg/kg po every 8 hours (maximum 3 g/day) from day +5 to day +35, and tacrolimus from day + 5 to day +180 to keep levels between 5 and 10ng/ml. Prophylactic antimicrobial therapy was started on day +1 and included fluconazole and acyclovir. ResultsFive children with AML and T-ALL (three in CR2; one induction failure and one 4th relapse) were offered haploidentical transplants as the first HSCT. They had a median age of 10 years (4-16) and weight 41 kg (14-67), 2 girls. One graft was a peripheral blood stem cell collection per physician preference. The median total nucleated cell count was 12x10(8)/kg (10-27) with 10x10(6) CD34/kg (5-15). Mucositis was mild in three patients and none of them had sinusoidal obstruction syndrome. All patients engrafted after a median 14 days (13-17) but one of them, in 4th relapse of acute promyelocytic leukemia (APL), rejected the graft. Two of four evaluable patients had mild acute graft versus host disease (GVHD), and one mild chronic GVHD. The patient with APL died of two graft rejections on day+125, one T-ALL relapsed on day+180 and three children are disease-free with excellent clinical status one month to 1.7 years after transplant. We conclude that haploidentical HSCT with post-transplant cyclophosphamide is feasible to treat children without an available matched related or unrelated donor and should be prospectively studied. Disclosures:No relevant conflicts of interest to declare.

Diagnostic value of cerebrospinal fluid analysis in a population of dogs with suspected idiopathic epilepsy

Cerebrospinal fluid (CSF) analysis is commonly used in the diagnostic investigation of seizure disorders in order to exclude possible inflammatory underlying aetiology. The medical records were searched for dogs presenting...

Family Mismatched Donor Transplantation for Myelofibrosis: A Retrospective Analysis of the EBMT Chronic Leukaemia Working Party

Background: Allogeneic stem cell transplantation is a treatment option for patients with advanced myelofibrosis. Problems encountered include an increased risk of delayed or poor engraftment and in the mismatched unrelated donor setting a higher rate of GVHD and particularly poor outcomes. As for other indications for allogeneic stem cell transplants, patients for whom either a matched sibling or matched unrelated donor is not available are considered for either a double umbilical cord blood, a mismatched unrelated donor or haploidentical stem cell transplant. Data on the latter option are limited and we reviewed registry data on all family mismatched donor transplants performed between 2001 and 2015 and reported to the EBMT registry.Results: Records retrieved 69 patients with myelofibrosis transplanted between November 2001 and November 2015. 44 (64%) were male. 50 (74%) had primary myelofibrosis,18 (27%) had secondary myelofibrosis (6 from ET, 5 from PRV and 7 others) and unknown 1(2%). Of 25 patients for whom the JAK2 V617F mutation status was known, 15 (22%) harboured the mutation. Patient Karnofsky performance status was > 70% in 98%.Of the mismatched family donors, 47 (68%) were male and 22 (31%) female. Donors were HLA mismatched at 1 locus in 12 (17%) and 2 or more loci in 48 (69%). Donor-recipient serology combinations were CMV -/- in 8 (12%), +/- in 4 (6%), -/+ in 15 (22%) and +/+ in 34 (49%) missing 8 (12%).Bone marrow was the stem cell source in 34 (49%) and peripheral blood in 35 (51%). The median total nucleated cell count (TNC) infused was 7.5x108/kg (range 2.3-21x108/kg) from data available in 17 patients. The median CD34+ cell dose was 6.9x106/kg (range 1.9-18.18x106/kg) from data available in 19 patients.Conditioning was myeloablative in 48 (70%) and RIC in 21 (30%) The conditioning regimes were varied but the predominant ones were Fludarabine, Busulphan, ATG (FBATG) and Thiotepa, Busulphan, Fludarabine (TBF). TBI was administered in 8 (12%) and in vivo T cell depletion in 22 (32%), ex-vivo T cell depletion in 5 (7%) patients. GVHD prophylaxis varied with post transplant Cyclophosphamide administered in 34/67 (49%) and ATG in 19/67 patients (28%).Neutrophil engraftment was established in 53 (82%) at a median of 20 days (range 11-83). Primary graft failure occurred in 8 (12%) and secondary graft failure in 4 (6%). This occurred at a median of 12 months (range 4.5-35 months). Eleven of these patients had a second allograft at a mean interval of 6.4 months. Responses to the first allograft (censoring for patients who had a second allograft) with data available in 45 patients, showed that complete remission was achieved in 35 patients (78%), 6 (13%) were never in CR and 4 (9%) were not evaluable.The cumulative incidence of grade II-IV acute GvHD at 100 days was 12% (95% CI 4-21%) and for grade III-IV acute GvHD at 100 days it was 5% (95% CI 3-11%). Data for chronic GVHD was valid in 49 patients. The cumulative incidence of chronic GvHD at 2 years was 62% (95% CI 47-76%). The cumulative incidence of limited cGvHD was 45% (95% CI 31-59%) whereas the cumulative incidence of extensive cGvHD was 10% (95% CI 2-19%).The median follow up was 24 months (95% CI 13-35 months). The 2-year OS was 51% (95% CI 37-76%) and the 5-year OS was 38% (95% CI 21-55%). The 2-year RFS was 44% (95% CI 30-59%) and the 5-year RFS was 31% (95% CI 15-48%). The 2 year cumulative incidence of relapse was 14% (95% CI 5-24%). The 2 year NRM was 41% (95% CI 28-55%), which increased to 54% (95% CI 37-72%) at 5 years.The main causes of death were infection (16, 24%), GVHD (7, 10%) organ damage or failure (3, 5%), relapse/disease progression (1, 2%) and secondary malignancy or PTLD (1, 2%). On univariate analysis there was no significant effect of recipient gender, donor gender, degree of HLA mismatch 1 vs >1 Ag MM, CMV matching between donor and recipient, primary or secondary MF, disease stage at transplant (chronic versus advanced phase), conditioning intensity, conditioning regimen, GVHD prophylaxis with ATG or post transplant cyclophosphamide or stem cell source on overall survival.Conclusion: Concerns regarding engraftment and secondary graft failure have excluded patients with myelofibrosis from clinical trials of mismatched related donor transplant. The data suggest that engraftment is feasible, and PFS and OS can be attained with limited severe chronic GVHD with family mismatched donors in this setting. DisclosuresCiceri:MolMed SpA: Consultancy.

Efficacy of glucocorticoid monotherapy for treatment of canine meningoencephalomyelitis of unknown etiology: a prospective study in 16 dogs.

Canine non‐infectious, inflammatory meningoencephalomyelitis is termed meningoencephalomyelitis of unknown etiology (MUE) and may affect dogs of any age, breed or gender. Treatment with immunosuppressive medication has been widely reported, however no prospective clinical trials with a standard glucocorticoid monotherapy are available. The objectives were to compare the cerebrospinal fluid (CSF) analysis at diagnosis and after treatment with a standard glucocorticoid (GC) dose and to determine the survival time in dogs with MUE. We hypothesized that abnormal CSF findings would normalize in dogs with MUE, and survival time would be longer than previously reported for glucocortocoid therapy alone. Inclusion criteria were: (1) normal minimum database, (2) no GC use within 5 days, (3) magnetic resonance imaging performed, (4) negative infectious disease titres, and (5) abnormal CSF analysis. All dogs received GC therapy at 1 mg/kg per os q 12 h. Responders had normal CSF analysis at 1 month. Sixteen dogs met the inclusion criteria. Median total nucleated cell count (TNCC) and protein concentration at time of diagnosis were 39 cells/μL (0–1400 cells/μL), and 49 mg/dL (25–293 mg/dL), respectively. Median TNCC and protein concentration at 1 month were 1 cell/μL (0–120 cells/μL), and 24 mg/dL (13–175 mg/dL), respectively. Seven of 16 dogs (44%) were responders. There was no significant difference in survival between the CSF responders and CSF non‐responders (P = 0.85). Overall median survival was 602 days (45–654 days). This study supports using GC therapy in dogs with MUE.

Longer Procedure Duration and Large Volume Harvests Reduce the Likelihood of Achieving the Requested Cell Dose from Unrelated Bone Marrow Donors

IntroductionThe factors influencing the quality of bone marrow (BM) harvests and total nucleated cell count (TNC) achieved are poorly understood. As the number of BM harvest procedures falls (due to an increasing proportion of harvest requests for peripheral blood stem cells), concerns are raised around maintaining expertise in this area. We investigate, in four Anthony Nolan collection centers, the procedure- and donor -related factors influencing the quality of harvests, and likelihood of achieving the requested TNC.Materials & Method110 consecutive BM harvests of healthy unrelated donors (UD)s were performed according to standard Anthony Nolan protocols. Harvest quality was defined as TNC/ml volume collected. Univariate analyses of factors influencing the TNC and harvest quality were performed using Chi squared or Fisher’s test where appropriate.Results110 BM harvests were performed in 80 male and 30 female donors. The median donor age was 28 years (range 18-56 years).The median TNC requested was 4x108/kg, and median harvested TNC 4.2x108/kg recipient weight, with the requested TNC dose achieved in 50% of harvests. Higher volume harvests (greater than the median volume 1200 mls) were significantly less likely to achieve TNC> 4x108/kg recipient weight (38% vs 70% p=0.001), as were harvests with a procedure time greater than 30 minutes (p=0.037). We investigated donor factors influencing the harvest yield and found that donor/recipient weight discrepancy significantly impacted the TNC achieved: with only 18% of BM harvests from donors who weighed less than their recipient achieving TNC >4x108/kg compared to 62% harvests from donors who were heavier than their recipient (p=0.001). No other donor factor had a significant influence on the TNC.The median TNC/ml collected was 18.2x10^6/ml and harvests in donors>40 years were more likely to be of lower quality (p=0.036). No other donor factors affected harvest quality, however a significant variation between the 4 collection centers was observed: both the harvest quality (p=0.014) and volume collected (p=0.035) varied significantly between centers.ConclusionsDonor weight is a strong determinant of bone marrow harvest yield; we recommend that when multiple matched UDs are available, a donor with high body weight should be selected for BM harvest. Procedure factors including large volume harvests and long procedure times reduce the likelihood of achieving the requested TNC, and significant variation was seen between centers. Given the declining number of requests for BM harvests, it is crucial these are performed by the most experienced centers and that systems exist to ensure operators retain their expertise. DisclosuresNo relevant conflicts of interest to declare.

Comparison of bronchoalveolar lavage fluid obtained by manual aspiration with a handheld syringe with that obtained by automated suction pump aspiration from healthy dogs

To compare bronchoalveolar lavage (BAL) fluid obtained by manual aspiration (MA) with a handheld syringe with that obtained by suction pump aspiration (SPA) in healthy dogs. 13 adult Beagles. Each dog was anesthetized and bronchoscopic BAL was performed. The MA technique was accomplished with a 35-mL syringe attached to the bronchoscope biopsy channel. The SPA technique was achieved with negative pressure (5 kPa) applied to the bronchoscope suction valve with a disposable suction trap. Both aspiration techniques were performed in each dog in randomized order on opposite caudal lung lobes. Two 1 mL/kg aliquots of warm saline (0.9% NaCl) solution were infused per site. For each BAL fluid sample, the percentage of retrieved fluid was calculated, the total nucleated cell count (TNCC) and differential cell count were determined, and semiquantitative assessment of slide quality was performed. Comparisons were made between MA and SPA techniques for each outcome. 1 dog was removed from the study because of illness. The mean percentage of fluid retrieved (mean difference, 23%) and median TNCC (median distribution of differences, 100 cells/μL) for samples obtained by SPA were significantly greater than those for samples obtained by MA. In healthy dogs, BAL by SPA resulted in a significantly higher percentage of fluid retrieval and samples with a higher TNCC than did MA. Further evaluation of aspiration techniques in dogs with respiratory tract disease is required to assess whether SPA improves the diagnostic yield of BAL samples.

What Clinical Characteristics of Mothers and Babies Favorably Influence Cord Blood Potency? Defining Parameters That Can Guide Public Cord Blood Collection

Abstract 484 Background:Cord blood (CB) is a rich source of hematopoietic stem cells and provides access to transplantation for many patients who otherwise would not have a suitably matched donor. Numerous international public CB banks are actively accruing units to the public inventory. While public banking is an activity that benefits many patients by increasing access to hematopoietic stem cell transplantation, it is financially challenging to maintain operations of a public bank which must front all costs of inventory building and overall operations. It is well recognized that total nucleated cell (TNC) content as well as numbers of CD34+ and colony forming unit (CFU) cells define a superior cord blood unit (CBU). In the US, criteria defining a higher quality CBU have been established through the C.W. Bill Young Cell Transplantation Program’s National Cord Blood Inventory. Applying current criteria, only ∼40% of collected CBUs meet criteria for banking. To improve the yield of “bankable” CBUs and allow for better focus of resources, we hypothesized that certain maternal/donor characteristics may be predictive of TNC (also, CD34+ or CFU) and, therefore, be more likely to meet banking criteria. Methods:Between 9/07–7/09, CBUs donated to the Carolinas Cord Blood Bank (n=5282) and meeting criteria for public banking (n=5268) or identified for directed donation (n=14) were analyzed. Standard criteria for donor eligibility were followed, including gestational age >34 weeks, uncomplicated pregnancy and delivery, singleton birth, and maternal age ≥18 years. Technical characteristics were obtained as part of routine banking procedures (including post-processing TNC, CD34+ and CFU) and were correlated with clinical features of the mother and baby including maternal age, delivery type, baby gender, birth weight (BW), gestational age, race/ethnicity and collected CB volume. Univariate and multivariate logistic regression models were created dichotomizing for TNC, CD34+ and CFU content. Univariate and adjusted odds ratios are provided. Results:In this study cohort, the infant donors had a median gestational age and BW of 39.0 weeks (range, 34–42 weeks) and 3580g (range, 1800–5724g), respectively. Both genders were evenly represented although there were more Caucasian infants (58.9%) followed by African American (20.2%) and Hispanic (12.9%). Slightly more infants were born via vaginal delivery (59.8%) to mothers with a median age of 29 years (range, 18–53 years). The median TNC count was 11.7×108 (range, 2.9–55.5), CD34+ cells were 3.36×106 (range, 0.017–98.2) and CFU content was 34.0×105 (range, 0.6–193.3). In univariate analysis, maternal/donor characteristics that were associated with high TNC (defined as a TNC of >10×108) included Caucasian race [OR 1.29 (95%CI 1.14–1.46) p<0.0001], Caesarian delivery [OR 1.23 (95%CI 1.09–1.39) p<0.0001], higher gestational age [OR 1.16 (95%CI 1.10–1.22) p<0.0001] and BW>3500g [OR 1.86 (1.64–2.09) p<0.0001]. Conversely, African American race was a negative predictor of high TNC [OR 0.76 (95%CI 0.66–0.88) p=0.0002] while gender and Hispanic race were not significant. In multivariate modeling of TNC including maternal/donor characteristics, BW [>3500g; OR 1.75(95%CI 1.54–1.98) p<0.0001] was the best predictor of a high TNC followed by Caucasian race [OR 1.20(95%CI 1.06–1.36) p=0.004] and Caesarian delivery [OR 1.20 (95%CI 1.05–1.36) p=0.006). We also examined clinical variables predictive of a high CD34+ content (defined as >3×106). In univariate analysis, male gender [OR 1.31 (95%CI 1.19–1.49) p<0.0001], BW>4000g [OR 1.93 (95%CI 1.66–2.25) p<0.0001], older gestational age [(OR 1.19 (95% CI 1.14–1.25) p<0.0001] and Caucasian race [OR 1.18 (95%CI 1.06–1.32) p=0.003] were predictive of high CD34+ content. Interestingly, neither maternal nor donor characteristics were associated with high CFU growth in univariate or multivariate analysis. Conclusions:Maternal/donor characteristics are predictive of TNC and CD34+ in collected CBUs. However, no clinical characteristic predicted graft CFU content, the primary determinant of CBU potency. These associations could be used to prioritize donations, optimizing resource utilization and financial modeling. Of note, African American race was associated with lower TNC indicating that increased numbers of African American CBUs will need to be collected to achieve a robust inventory of higher quality units. Disclosures:No relevant conflicts of interest to declare.

Comparison of Pre-Cryopreserved and Post Thaw and Wash Total Nucleated Cell Count On Major Outcomes Following Unrelated Cord Blood Transplant in Children.

Abstract 3336Poster Board III-224Engraftment and overall survival after umbilical cord blood transplant is highly dependent on the total nucleated cell count (TNC). Current standard post thaw processing includes a wash step to remove dimethyl sulfoxide (DMSO), lysed red cells and stroma. The contribution of the wash step to cell loss and ultimately the dose of cells available for transplant is not well described. To investigate the amount of cell loss after washing and its impact on major outcomes compared to pre-cryopreserved TNC, we analyzed data from 310 patients prospectively enrolled on a National Heart Lung Blood Institute (NHLBI) sponsored cord blood transplant study between 1999 and 2003. Dataset was obtained after signed agreement with the NHLBI and local IRB approval. There were 310 patients ≤18 years of age with malignant (N=217) or non-malignant (N=93) disease enrolled on this trial. Only single cord units were used. All cord blood units were thawed and washed using an identical process developed by Rubinstein et al. All patients received myeloablative preparative regimen with either total body irradiation or busulfan based regimens with cyclosporine and prednisone GVHD prophylaxis. All patients received anti-thymocyte globulin as part of their conditioning regimen. For the overall survival, Cox proportional hazard models were generated for pre-wash cell dose and post-wash cell dose separately and then combined in one model. All models included identical covariates. Total cell dose was modeled as a continuous variable with appropriate transformation using restricted cubic lines to account for non-linear relationships. For transplant related mortality (TRM) and neutrophil engraftment, competing risk analyses were used. These analyses were done with adjustment for age, gender, disease (malignant versus nonmalignant), performance status (<90 versus ≥90), HLA (3-4/6 versus 5-6/6 match), and CMV status. The median age was 4.59 years (range 0.04 – 17.90) with 188 (61%) male, 249 patients (80%) had a performance status of ≥90. 166 patients (54%) received a cord blood unit matched at 3/6 or 4/6 HLA antigens and 144 patients (46%) received a cord blood unit matched at 5/6 or 6/6 HLA antigens. The median pre-cryopreserved TNC per kg was 6.93 × 107/kg (range 1.5-80.9 × 107/kg). The median TNC recovery after thawing and washing (PTW) was 5.43 × 107/kg (range 1-31.6 × 107/kg). The average cell recovery was 89% after thawing and washing. Neutrophil engraftment was significantly associated with higher pre-cryopreserved (p=0,003) and PTW TNC infused (p=0.005); younger age (p=0.03), better HLA match (p=0.03). The risk of transplant related mortality was significantly higher among older patients (p=0.02), female patients (p=0.02) and those receiving 3-4/6 HLA matched cord units (p=0.02). Neither the pre-cryopreserved or PTW TNC were significant contributing factors. The risk of grade II-IV acute GVHD was significantly higher among older patients (p=0.04) and those receiving higher pre-cryopreserved TNC (p=0.02) but not higher PTW TNC (p=0.07). Overall survival was significantly better among younger patients (p=0.02), male recipients (p<0.001), patients with non-malignant diseases (p<0.001), patients with performance status >90 (p=0.04) and those receiving 5-6/6 HLA matched cord units (p=0.04). Pre-cryopreserved and PTW TNC did not influence overall survival. In conclusion, pre-cryopreserved and post thaw and wash TNC were equally predictive for major outcomes of unrelated cord blood transplant in children. DisclosuresNo relevant conflicts of interest to declare.

The Effect of Obesity On Outcome of Unrelated Cord Blood Transplant in Children with Malignant Diseases.

Abstract 2278Poster Board II-255Obesity has become a pandemic in the US, affecting both children and adults. In the adult population, evidence suggests that obesity has an adverse impact on outcomes of hematopoietic stem cell transplant for various malignancies. We investigated the influence of obesity on children undergoing unrelated cord blood transplant utilizing data from the Cord Blood Transplant Study. To our knowledge, no data exists on the effect obesity has on key prognostic indicators for cord blood transplant for malignant diseases in children. There were 191 patients '18 years of age with malignant diseases prospectively enrolled on a National Heart Lung Blood Institute (NHLBI) sponsored cord blood transplant study between 1999 and 2003. Data set was obtained after signed agreement with the NHLBI and local IRB approval. All patients received myeloablative preparative regimens with either total body irradiation and cyclophosphamide (N=172) or busulfan and melphalan (N=19). In addition, all patients received anti-thymocyte globulin as part of their conditioning regimens. Graft versus host disease (GVHD) prophylaxis included cyclosporine and prednisone. Children were classified into groups according to body mass index (BMI) percentile. Normal weight was defined as BMI between the 5th and 85th percentile, overweight between the 85th and 95th percentile, obesity above 95th percentile for age and gender according to the Center for Disease Control and Prevention (CDC) guidelines. For the overall survival (OS) and disease free survival (DFS), the Cox regression model was used to test the effect of BMI while controlling for age, gender, performance status (<90 versus ≥90), HLA match (≤4/6 versus ≥5/6 HLA match), total nucleated cell count (TNC) per kg infused and CMV status. For neutrophil and platelet engraftment, transplant related mortality (TRM), grade II-IV acute GVHD, and chronic GVHD, competing risks regression analyses were used to test effect of BMI while controlling for other covariates. The median age was 7.59 years (range 2.07 – 17.90) with 113 (59%) male. 160 patients (84%) had a performance status of ≥90. 51 patients (27%) had acute myelogenous leukemia (AML), 109 patients (57%) had acute lymphoblastic leukemia (ALL) and 30 patients (16%) had other malignant diseases. 119 patients (62%) received a cord blood unit matched at 3/6 or 4/6 HLA antigens and 72 patients (38%) received a cord blood unit matched at 5/6 or 6/6 HLA antigens. The median TNC per kg infused was 5.2 × 107/kg (range 0.15-80.9 × 107/kg). Of the 191 total patients who were classified by their BMI percentiles, 117 patients (61%) were normal weight, 35 patients (18%) were overweight and 39 patients (20%) were obese. The two groups were not significantly different in sex distribution (p=0.25), diagnosis (p=0.13), performance status (p=0.91), median TNC received (p=0.49) or CMV status (p=0.47). Obese patients were significantly younger with a median age of 5.7 years compared to 8.9 years in normal weight children (p=0.002). Time to neutrophil and platelet engraftment, TRM, risk of grade II-IV acute GVHD, DFS and OS were not significantly different in overweight or obese patients compared to normal weight patients. There was a trend towards increased risk of chronic GVHD in obese patients (p=0.045) compared to normal weight patients. In conclusion, obesity has no significant effect on multiple outcomes after unrelated cord blood transplant in children with malignant diseases. Disclosures:No relevant conflicts of interest to declare.

High Rate of Engraftment and Low Regimen-Related Toxicity Using Fludarabine, Melphalan and Thiotepa Conditioning for Unrelated Donor Cord Blood Transplantation

BACKGROUND: Cord blood transplantation (CBT) represents an alternative source of stem cells for adult patients who lack a matched sibling or unrelated donor. However, the optimal type and intensity of the preparative regimen for patients receiving a CBT is not clear. We hypothesized that a conditioning regimen consisting of fludarabine, melphalan and thiotepa will be associated with an acceptable rate of engraftment and treatmentrelated mortality in patients receiving a CBT.METHODS: 37 patients, median age 31 years (2–57) and a median weight of 73kg, were treated between 8/2003 and 5/2008. All had advanced hematologic malignancies (24 with acute leukemia, 12 with lymphoma and one with CLL) At the time of transplant, 21 pts (57%) were in complete remission (CR) (first CR=20%) and 16 had relapsed/refractory disease. Grafts consisted of double (29 pts) or single (8 pts) CB units. Cytogenetics for patients with acute leukemia were poor in 11, intermediate in 9, good in 1 and unknown in 3 pts. Donor recipient HLA matching was (intermediate resolution class I HLA A and B and high-resolution DRB1): 3/6 (n=1, 1.5%), 4/6 (n=47, 71.2%) and 5/6 (n=18, 27.3%) alleles (n=66 units). Median total nucleated cell count was 1.8×107/kg (range 1–5.8). Nineteen patients received ex-vivo expanded units. The conditioning regimen consisted of melphalan 140 mg/m2 on day -8, thiotepa 10 mg/m2 on day -7, fludarabine 160 mg/m2 over 4 days on days -6, -5, -4, -3, and rabbit ATG 1.25 mg/kg on day -4 and 1.75 mg/kg on day -3 (FMT). Patients with CD 20+ lymphoid malignancies also received rituximab 375mg/m2 on day -9 (n=8, 22%). GVHD prophylaxis was tacrolimus and mini-methotrexate in 23 (62%) and tacrolimus and mycophenolate in 14 pts (38%).RESULTS: 36 patients (97%) were evaluable for engraftment. 1 patient died within 30 days due to progressive leukemia. 34/36 patients (95%) engrafted neutrophils and had hematopoietic recovery with 100% cord blood-derived cells. At day 30, of the 29 patients who received a double CBT, 75% had chimerism derived entirely from one donor while 25% had mixed donors chimerism. Neutrophil recovery to ANC >0.5 × 10e9/l occurred after a median of 21 days (range 6–45) and platelet recovery to >20 × 10e9/l after a median of 37 days (range 26–134, N=24; 67%). 32/37 pts (87%) were in CR after transplant with 16 surviving after a median follow-up of 12.1 months. Thirteen patients (36%) developed gr II–IV aGVHD (gr III–IV aGVHD in 5 patients, 14%), and 13 of 32 patients had cGVHD (40%), with the majority experiencing extensive GVHD. 11 patients (29.7%) relapsed after a median of 7 months post transplant and 12 died of nonrelapse causes. Day-100 treatment-related mortality in this heavily pre-treated population was 10%. Overall, causes of death included disease relapse (n=9), infections (n=6), organ failure (n=3), pulmonary hemorrhage (n=1) and GVHD (n=2).CONCLUSIONS: The FMT regimen was sufficiently immunosuppressive to support high rate of engraftment with acceptable TRM in heavily pre-treated adult patients with advanced hematologic malignancies undergoing CBT. These results support further evaluation of this regimen in CBT.

Reduced Intensity (RI) Allogeneic Cord Blood Hematopoietic Cell Transplantation (Allo CBHCT) in Pediatric Patients with Malignant and Non-Malignant Diseases.

We and others have demonstrated that cord blood hematopoietic cells can be used successfully as an alternative allogeneic progenitor cell source in pediatric and adult recipients with both malignant and non-malignant diseases (Cairo et al, Blood 1997). However, there is significant amount of morbidity and mortality following myeloablative CBHCT (Gluckman et al, NEJM 1997; Rubinstein et al, NEJM 1998). RI conditioning may potentially reduce the incidence of regimen-related mortality (RRM) and the late effects associated with myeloablative regimens without increasing the risk of graft failure or relapse in children and adolescents (Del Toro et al, BMT 2004). Successful engraftment has been demonstrated in adult recipients with advanced hematologic malignancies who received unrelated mismatched cord blood following RI conditioning regimens (Chao et al, BBMT 2004). We report the results in 22 pediatric recipients, 6F:16M, median age 6.5 years (0.4–21). Twenty-one received unrelated CBHC, the majority mismatched at 1–2 HLA-Loci, 4/6 (n=14), 5/6 (n=5), 6/6 (n=2); 1 sibling CBHC (6/6). Recipients were transplanted for malignant n=17, [HD (5), NHL (2), NB(4),AML (2), MDS(3),CML (1)] and non-malignant n=5, [b-thal (1), HLH (2), WAS (1), SCD(1)] diseases. Poor-risk disease was defined as induction failure, PD, SD, ≥CR3, and 2nd allo HCT (n=8). RI conditioning was fludarabine-based (150–180mg/m2) with either busulfan (8mg/kg) +/− rabbit ATG/alemtuzumab or cyclophosphamide +/− etoposide/etoposide phosphate +/− ATG and craniospinal radiation prophylaxis (n=1). Graft-vs.-host-disease (GHVD) prophylaxis: tacrolimus 0.03 mg/kg/day and mycophenolate mofetil 900mg/m2 q 6h (Osunkwo et al, BBMT 2004). Median total nucleated cell count was 3.8 x107/kg (0.92–10.82) and CD34 cell count was 2.2 x105/kg (0.34–6.95). The median day to neutrophil engraftment was 19.5 days (1–47) and to platelet engraftment was 40 days (6–170). Engraftment was 72.7%. There were 6 primary graft failures observed (1 CML, 1 b-Thal, 2 HLH, 1 MDS, and 1 AML). Within this subgroup of graft failures, 4 were regrafted with myeloablative conditioning followed by a second CBHCT and 100% engrafted. Of the 16 engrafted patients, 15 achieved 90% or greater mixed-donor chimerism by day 60. Five of 16 evaluable patients developed acute GVHD, with 3 of those 5 developing grade III/IV acute GVHD. One patient developed extensive chronic GVHD. There were 8 deaths (4 PD, 1RRM, 3 GVHD). The probability of 3-year overall survival (OS) in all patients was 61%. Despite a log less nucleated cells/kg with CB vs. PB or BM HCT, the 3-yr OS in standard-risk malignancy pts. was 90%. The 1-yr OS in poor-risk malignancy pts. was 21%. The 1-yr OS in non-malignant diseases was 75%.These preliminary results indicate that RI Allo CBHCT may result in more rapid hematopoietic reconstitution while decreasing RRM compared to myeloablative CBHCT and be associated with high-to-full mixed-donor chimerism. Patients with diseases such as CML, β-Thal, and HLH may require increased intensity of conditioning. Further follow-up is required to evaluate the difference, if any, in long-term effects following RI Allo CBHCT.

Higher Cell Dose and CD34+ Content Improves Engraftment Following Unrelated Donor Cord Blood Transplantation (CBT): A Report of the National Marrow Donor Program (NMDP) Cord Blood (CB) Experience.

Cord blood has become an important alternative unrelated donor allogeneic hematopoietic stem cell source. The NMDP has developed a comprehensive coordinated network of CB banks, search coordinating center and transplant programs with prospective collection of outcome data coordinated by the NMDP (now CIBMTR). Critical to CBT has been limited cell dose with resultant prolonged engraftment time. The NMDP cord blood inventory has both total nucleated cell count (TNC) and CD34+ quantification on the units, allowing a comparison of the relative utility of either measure in identifying units that would result in rapid engraftment. We report a retrospective review of the NMDP CBT experience, analyzing the impact of graft characteristics with engraftment and survival. Between 03/2000 and 03/2004, 12 NMDP banks (total inventory 31,976 units) released CB units to 144 patients at 44 NMDP transplant programs included in this analysis (median f/u 217 days, 26–1204 days). The median recipient age was 8.2 years (0.2–63.1 years, 38 were ≥ 15 years) and median weight was 27 kg (3–158 kg: 26% > 57 kg). Transplant indications included malignancy in 113 (ALL 36, AML 43, MDS 13, other 21), metabolic disorders (8) immune disorders (9) histiocytic disorders (3), erythrocytic abnormalities (6), platelet abnormality (1), SAA (3) and other nonmalignant disease (1). Most malignancy patients had advanced disease (60 patients (53%) were beyond CR2 or in relapse). The median prefreeze TNC was 4.4 x 107/kg (0.3–433 x 107/kg) and CD34+ cells 7.9 x 105/kg (1.1–68.5 x 105/kg) in units selected for transplantation. Thus the median CB TNC was 142 x 107 cells (54–396 x 107 cells); only 12 units under 80 x 107 cells were used. 114 patients engrafted by day +42 post CBT with median time to neutrophil recovery > 500/mm3 of 21 days (8–62 days) and platelet count > 20,000 x 109/L of 64 days (12–473) respectively. 1-year survival and DFS were 39% ± 9% and 38% ± 9% respectively. Relapse rate was 16% ± 8% in this high risk population. The 100-day TRM rate was 26% ± 7%. For patients > 15 yrs, TRM was 42% ± 16% vs. 21% ± 8% for patients < 15 years. Higher cell dose was associated with faster neutrophil and platelet engraftment. Units with both high TNC/kg and high CD34+/kg were associated with more rapid engraftment vs. those with only high TNC or high CD34+ or neither (p<0.0001). In multivariate analysis, recipient age > 15 years led to poorer survival (RR 3.4 (1.7, 6.7)) and DFS (RR 2.8 (1.5, 5.2)) compared to younger children, especially those < 3 years (p<0.0001). Male grafts into females yielded poorer survival than other gender combinations. These data confirm that CB is a valuable alternative unrelated donor histocompatible stem cell source. Since transplantation using CB units containing both high TNC and CD34+ content resulted in more rapid engraftment, optimal CB inventory should strive for both high cell count (> 80 x 107 cells) and high CD34+ cell content.

A single centre, randomised trial on harvest cell yield and marrow engraftment using haemopoietic growth-factor primed bone marrow

The use of haemopoietic growth factors (HGFs) in the BMT setting is most often administered to recipients during the post-BMT period to enhance neutrophil recovery. However, there is little or no effect on platelet recovery when HGFs are administered in this fashion. The aim of this study is to compare HGFs primed and unprimed marrow in terms of: the haemopoietic stem and progenitor cell yields; the neutrophil and platelet engraftment in allogeneic BMT; and the incidence and severity of GVHD as well as other complications in allogeneic BMT. Methods: Thirteen patient-donor pairs were randomized to the G-CSF group, eleven pairs to the GM-CSF group and thirteen pairs to the control group. The donors randomized to group I received 10μg/kg per day G-CSF subcutaneously for four days before the bone marrow was harvested. The donors randomized to group II received 10μg/kg per day GM-CSF subcutaneous injection for four days before the bone marrow was harvested. The donors randomized to the control arm did not receive any growth factors before the bone marrow was harvested. Results: The median total nucleated cell count was slightly higher in primed BM graft. For the G-CSF primed BM, 5.77 x 108/kg, and for the GM-CSF primed BM 3.18 x 108/kg compared to 2.23 x 108/kg for the unprimed BM graft (Kruskal Wallis test, P < 0.001). The central tendency was significantly different (Kruskal Wallis Test, P < 0.003) between the three groups for the CFU-GM/kg measure. The median was 17.44 x 104/kg in the G-CSF group, 6.14 x 104/kg in the GM-CSF group and 10.12 x 104/kg in the control group. The mean time to obtain an ANC value above 1.0 x 109/L was statistically significant among the three groups (ANOVA, P < 0.046); the mean duration for group I, II and III was 23 days, 30 days and 29 days, respectively. We found that G-CSF primed BM had significantly faster engraftment of ANC > 1.0 x 109/L, compared to GM-CSF primed BM and unprimed BM. Besides, the units of RBC used post BMT were significantly less than the other two groups. G-CSF priming increased total nucleated cells and proliferation of CFU-GM in the marrow graft.