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Background

We evaluated 26 901 patients who underwent allogeneic hematopoietic cell transplantation (HCT) at 271 centers worldwide to define patterns of posttransplantation lymphoproliferative disorders (PTLDs). PTLDs developed in 127 recipients, with 105 (83%) cases occurring within 1 year after transplantation. In multivariate analyses, we confirmed that PTLD risks were strongly associated (P < .001) with T-cell depletion of the donor marrow, antithymocyte globulin (ATG) use, and unrelated or HLA-mismatched grafts (URD/HLA mismatch). Significant associations were also confirmed for acute and chronic graft-versus-host disease. The increased risk associated with URD/HLA-mismatched donors (RR = 3.8) was limited to patients with T-cell depletion or ATG use (P = .004). New findings were elevated risks for age 50 years or older at transplantation (RR = 5.1; P < .001) and second transplantation (RR = 3.5; P < .001). Lower risks were found for T-cell depletion methods that remove both T and B cells (alemtuzumab and elutriation, RR = 3.1; P = .025) compared with other methods (RR = 9.4; P = .005 for difference). The cumulative incidence of PTLDs was low (0.2%) among 21 686 patients with no major risk factors, but increased to 1.1%, 3.6%, and 8.1% with 1, 2, and more than 3 major risk factors, respectively. Our findings identify subgroups of patients who underwent allogeneic HCT at elevated risk of PTLDs for whom prospective monitoring of Epstein-Barr virus activation and early treatment intervention may be particularly beneficial.

The apoptotic and therapeutic activities of the histone deacetylase inhibitor (HDACi) vorinostat are blocked by overexpresssion of Bcl-2 or Bcl-XL. Herein, we used the small molecule inhibitor ABT-737 to restore sensitivity of Eμ-myc lymphomas overexpressing Bcl-2 or Bcl-XL to vorinostat and valproic acid (VPA). Combining low-dose ABT-737 with vorinostat or VPA resulted in synergistic apoptosis of these cells. ABT-737 was ineffective against Eμ-myc/Mcl-1 and Eμ-myc/A1 cells either as a single agent or in combination with HDACi. However, in contrast to the reported binding specificity data, Eμ-myc/Bcl-w lymphomas were insensitive to ABT-737 used alone or in combination with HDACi, indicating that the regulatory activity of ABT-737 is restricted to Bcl-2 and Bcl-XL. Eμ-myc lymphomas that expressed Bcl-2 throughout the tumorigenesis process were especially sensitive to ABT-737, while those forced to overexpress Mcl-1 were not. This supports the notion that tumor cells “addicted” to ABT-737 target proteins (ie, Bcl-2 or Bcl-XL) are likely to be the most sensitive target cell population. Our studies provide important preclinical data on the binding specificity of ABT-737 and its usefulness against primary hematologic malignancies when used as a single agent and in combination with HDACi.

Introduction

Introduction:

Follicular lymphoma is a rare lymphoid malignancy in pediatric patients and consequently remains poorly characterized, particularly with respect to its immunophenotype and molecular pathogenesis. A total of 23 pediatric patients with follicular lymphoma were identified, with a median age of 11 years and a male-to-female ratio of 2.3:1. Of the 19 patients for whom presenting clinical features were available, 15 patients had stage I, 1 had stage II, and 3 had stage III or IV disease. All tumors had a follicular architecture, and 74% of cases had grade 2 or 3 histologic features. All patients expressed CD20 and bcl-6, and 80% were positive for CD10. Bcl-2 expression was detected in only 5 of 16 cases. Consistent with this finding, bcl-2 gene rearrangements were detected in only 2 of 16 cases by polymerase chain reaction. These patients were treated primarily with cyclophosphamide, doxorubicin, vincristine, and prednisone–based chemotherapy; 4 patients also received involved-field irradiation. Of the 13 patients with available clinical follow-up, all but 2 achieved durable clinical remission. Importantly, all 4 patients with tumors diffusely positive for bcl-2 either presented with stage III/IV disease or had disease refractory to therapy, whereas patients with bcl-2–negative tumors uniformly had stage I disease, achieved complete remission, and experienced no relapses. These findings indicate that, in contrast to adult follicular lymphomas, dysregulated bcl-2 expression does not play a significant pathogenetic role in most pediatric follicular lymphomas. However, bcl-2 expression in pediatric follicular lymphoma identifies a subset of patients in whom disease is often disseminated at clinical presentation and is more refractory to combination chemotherapy.

Background:Pevonedistat is a first-in-class inhibitor of NEDD8-activating enzyme that catalyzes the rate-limiting step of protein neddylation, a critical step in the degradation of cellular proteins that occurs upstream of the proteasome. The combination of azacitidine plus pevonedistat has resulted in high response rates and durable remissions in both myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), particularly in secondary AML (s-AML). Preclinical studies suggest that pevonedistat synergizes with venetoclax through neutralization of Mcl-1, providing rationale for the triplet combination of azacitidine, venetoclax and pevonedistat.

Background:Blinatumomab is highly effective therapy for both the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and persistent or recurrent measurable residual disease (MRD) after initial ALL therapy. We hypothesized that early incorporation of blinatumomab in patients (pts) with newly diagnosed Philadelphia chromosome (Ph)-negative B-cell ALL would lead to deeper and more durable responses, reduce relapses, and improve survival.

We evaluated 18,014 patients who underwent allogeneic bone marrow transplantation (BMT) at 235 centers worldwide to examine the incidence of and risk factors for posttransplant lymphoproliferative disorders (PTLD). PTLD developed in 78 recipients, with 64 cases occurring less than 1 year after transplantation. The cumulative incidence of PTLD was 1.0% ± 0.3% at 10 years. Incidence was highest 1 to 5 months posttransplant (120 cases/10,000 patients/yr) followed by a steep decline to less than 5/10,000/yr among ≥1-year survivors. In multivariate analyses, risk of early-onset PTLD (<1 year) was strongly associated (P < .0001) with unrelated or human leukocyte antigen (HLA) mismatched related donor (relative risk [RR] = 4.1), T-cell depletion of donor marrow (RR = 12.7), and use of antithymocyte globulin (RR = 6.4) or anti-CD3 monoclonal antibody (RR = 43.2) for prophylaxis or treatment of acute graft-versus-host disease (GVHD). There was a weaker association with the occurrence of acute GVHD grades II to IV (RR = 1.9, P = .02) and with conditioning regimens that included radiation (RR = 2.9, P = .02). Methods of T-cell depletion that selectively targeted T cells or T plus natural killer (NK) cells were associated with markedly higher risks of PTLD than methods that removed both T and B cells, such as the CAMPATH-1 monoclonal antibody or elutriation (P = .009). The only risk factor identified for late-onset PTLD was extensive chronic GVHD (RR = 4.0, P = .01). Rates of PTLD among patients with 2 or ≥3 major risk factors were 8.0% ± 2.9% and 22% ± 17.9%, respectively. We conclude that factors associated with altered immunity and T-cell regulatory mechanisms are predictors of both early- and late-onset PTLD.

To establish the clonal origin of a case of concomitant B-cell chronic lymphocytic leukemia (IgM kappa) and multiple myeloma (IGA lambda), we analyzed the immunoglobulin (Ig) gene rearrangements in the patient's blood and bone marrow. Despite the different isotypes, pretreatment investigation of the heavy chain gene (JH) revealed a germline fragment and two identical rearrangements in the blood and marrow. Both kappa and lambda light-chain genes were rearranged in the blood, suggesting peripheral blood lymphocyte involvement in the myeloma. Analysis of the Ig genes after chemotherapy demonstrated no change in the JH or CK rearrangements; however, the lambda genes were now in a germline configuration. Our results suggest that in this patient both chronic lymphocytic leukemia and myeloma originated from the same B-cell progenitor.

Effects of soluble recombinant human type I interleukin-1 receptor (sIL-1RI) were evaluated in 18 volunteers given intravenous endotoxin and randomized to placebo (n = 6), low-dose (n = 6), or high-dose (n = 6) sIL-1RI. Soluble IL-1RI decreased IL-1 beta (P = .001), but decreased IL-1ra (P = .0001), and resulted in 10-fold and 43-fold dose-related increases in sIL-1RI-IL-1ra complexes compared with placebo (P < or = .001). High-dose sIL-1RI was associated with increased levels of immunoactive tumor necrosis factor-alpha (P = .02), IL-8 (P = .0001), and cell-associated IL-1 beta (P = .047). C-reactive protein levels were higher after sIL-1RI than placebo (P = .035). Soluble IL-1RI decreased the severity of chills (P = .03), but did not alter other symptoms, changes in temperature, systemic hemodynamic responses, or changes in leukocyte and platelet number. Thus, sIL-1RI had no discernable antiinflammatory effect following endotoxin administration due in part to low levels of circulating IL-1 beta and neutralization of IL-1ra inhibitory function. This latter interaction represents an indirect mechanism of agonist activity elicited by sIL-1RI and may contribute to increases in inflammatory mediators, limiting therapy with sIL-1RI during endotoxemia.

The objectives of this study were (1) to determine the clinical presentation and natural history associated with two newly recognized pathologic entities termed mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL), including the mucosa-associated lymphoid tissue (MALT) and monocytoid B-cell subcategories, and (2) to determine whether these entities differ clinically from the other relatively indolent non- Hodgkin's lymphomas with which they have been previously classified. We reviewed the conventional pathology and clinical course of 376 patients who had no prior therapy; had stage III/IV disease; were classified as Working Formulation categories A, B, C, D, or E; and received cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) on Southwest Oncology Group (SWOG) studies no. 7204, 7426, or 7713. All slides were reviewed by the three pathologists who reached a consensus diagnosis. Age, sex, performance status, bone marrow and/or gastrointestinal involvement, failure-free survival, and overall survival were compared among all the categories. We found that (1) MCL and MZL each represent approximately 10% of stage III or IV patients previously classified as Working Formulation categories A through E and treated with CHOP on SWOG clinical trials; (2) the failure-free survival and overall survival of patients with MZL is the same as that of patients with Working Formulation categories A through E, but the failure-free survival and overall survival of the monocytoid B-cell patients were higher than that of the MALT lymphoma patients (P = .009 and .007, respectively); and (3) the failure-free survival and overall survival of patients with MCL is significantly worse than that of patients with Working Formulation categories A through E (P = .0002 and .0001, respectively). In conclusion, patients with advanced stage MALT lymphomas may have a more aggressive course than previously recognized. Patients with MCL do not have an indolent lymphoma and are candidates for innovative therapy.

Lymph nodes from each of the four histologic types of Hodgkin's disease were examined for the presence of eosinophils and for eosinophil degranulation by immunofluorescent localization of eosinophil granule major basic protein (MBP). Eosinophil degranulation shown by MBP deposition outside of eosinophils was found in six of eight nodes from patients with nodular sclerosing disease and in two of eight nodes from patients with lymphocyte depletion-type disease. Three nodes of the mixed cellularity type, one node of the lymphocyte predominance type, and one lymph node of the lymphocyte depletion type showed one or two small foci of extracellular MBP deposition. Lymph nodes from patients without Hodgkin's disease showed no extracellular deposition of MBP. Large numbers of eosinophils were found in seven of eight nodes of the nodular sclerosing variant, but were less frequently seen among the other types of Hodgkin's disease. The presence of extracellular MBP in lymph nodes of patients with Hodgkin's disease indicates that eosinophil degranulation commonly occurs and suggests that the released eosinophil granule proteins may participate in the inflammatory reaction in this disorder more extensively than is presently appreciated.

To establish the clonal origin of a case of concomitant B-cell chronic lymphocytic leukemia (IgMκ) and multiple myeloma (IGAX), we analyzed the immunoglobulin (Ig) gene rearrangements in the patient's blood and bone marrow. Despite the different isotypes, pretreatment investigation of the heavy chain gene (JH) revealed a germline fragment and two identical rearrangements in the blood and marrow. Both Kand X light-chain genes were rearranged in the blood, suggesting peripheral blood lymphocyte involvement in the myeloma. Analysis of the Ig genes after chemotherapy demonstrated no change in the JHor CKrearrangements; however, the X genes were now in a germline configuration. Our results suggest that in this patient both chronic lymphocytic leukemia and myeloma originated from the same B-cell progenitor.

The purpose of this study was to determine the relative merits of idarubicin and daunorubicin in acute myeloid leukemia (AML) therapy. Thirty-two sites provided 214 previously untreated adults with AML aged 15 years or more who were randomized to receive for induction therapy cytarabine 100 mg/m2/d as a continuous 7-day infusion plus either daunorubicin 45 mg/m2/d (A + D) or idarubicin 13 mg/m2/d (A + I), daily on the first three days of treatment. Postremission therapy consisted of two courses of the induction regimen at the same daily doses, with the anthracycline administered for 2 days and cytarabine for 5. The complete response (CR) rates for evaluable patients were 70% (A + I) and 59% (A + D) (P = .08). The difference in CR rates was significant in patients aged 18 to 50 years (88% for A + I, 70% for A + D, P = .035). Resistant disease was a significantly more frequent cause of induction therapy failure with A + D than with A + I. Hyperleukocytosis (white blood cell count greater than 50,000/microL) unfavorably affected the attainment of CR with A + D but not with A + I. CR duration was significantly greater after A + I. CR duration was significantly greater after A + I treatment, and the survival of all randomized patients treated with A + I was significantly better than that observed after A + D treatment (median 12.9 months v 8.7 months, respectively, P = .038). Toxicity of the two treatments was similar, although A + I patients experienced more prolonged myelosuppression during consolidation therapy, and a greater incidence of mild chemical hepatitis was observed in the A + I group. It is concluded that, at the doses and schedule used in this study, A + I is superior to A + D for induction therapy of AML in adults.

Fifteen of 52 patients (29%) with diffuse histiocytic and undifferentiated pleomorphic lymphoma developed central nervous system (CNS) complications, primarily leptomeningeal lymphoma. Lumbar puncture with cerebrospinal fluid cytology was the most useful test for diagnosis, and for following the response to therapy. Leptomeningitis developed during all stages of the patients’ clinical course: at time of diagnosis, during progression of systemic disease, and most importantly as the initial site of relapse within 7 mo of attaining a complete clinical remission. Patients with bone marrow involvement are at high risk for the development of leptomeningeal lymphoma. Pathologic findings suggest that entry into the leptomeninges involves extension from the medullary bone marrow cavity along perforating vessels through dura into the arachnoid space. The leptomeningeal lymphoma has been successfully controlled in all patients receiving intensive central nervous system therapy consisting of a combination of intrathecal drug administration and radiotherapy. The high frequency of this syndrome and the success in its control suggest that a controlled trial of prophylactic CNS therapy be instituted in patients with these histologic types of non-Hodgkin’s lymphomas.

One hundred twenty-five cases of Hodgkin's disease from the United States (79), Mexico City (31), and Costa Rica (15) were analyzed for the presence of Epstein-Barr virus (EBV) by in situ hybridization to EBER1 transcripts. EBV was more frequently detected in the Reed- Sternberg (RS) cells of mixed cellularity Hodgkin's disease (37 of 48 [77%]) compared with the nodular sclerosis subtype (19 of 71 [27%], P < .001). The presence of EBV was also associated with Hispanic ethnicity (P < .001). In a multivariate analysis, patient age, gender, and geographic location were less predictive of EBV positivity than were mixed cellularity histology (odds ratio = 8.3) and Hispanic ethnicity (odds ratio = 4.3). Southern blot analysis of EBV terminal repeat fragments using the Xho1a probe showed that the viral DNA was monoclonal in 17 of 17 cases having EBER1-positive RS cells. By comparison, EBV DNA was not detected by Southern analysis in 20 cases lacking EBER1 in RS cells, even when occasional background lymphocytes expressed EBER1. Because clonal viral DNA was so readily detected in EBER1-positive cases, the EBV genome is probably amplified at least 50- fold in the infected RS cells. Monoclonality of EBV DNA implies that the RS cells were infected before malignant transformation.

The growth fraction of tumors from patients with non-Hodgkin's lymphomas (NHL) has been shown to correlate with survival in retrospective studies. The growth fraction can be evaluated using immunohistochemical techniques employing the Ki-67 monoclonal antibody (MoAb) that marks a nuclear protein present in cycling cells. The purpose of this study was to evaluate the clinical utility of the Ki-67 MoAb for predicting survival. Using a prospective trial design in a multi-institutional cooperative trials group, the proliferative index, clinical outcome, and statistical correlations were independently assessed for previously untreated patients with advanced stages of intermediate- and high-grade histologies of NHL treated on Southwest Oncology Group study (SWOG 8516, Intergroup 0067). The proportion of Ki- 67-positive cells was determined on snap-frozen thin tissue sections. A proliferative index of 80% or greater, as determined from prior retrospective studies, identified a group of patients (18%) who had a poor outcome. Overall survival was significantly reduced in these patients with a high Ki-67-associated proliferative index compared with those with a low proliferative index (P = .001). One-year survival estimates were 82% (low proliferative index) versus 18% (high proliferative index). A multivariate regression analysis incorporating commonly used clinical prognostic features confirmed the independent effect of proliferation on survival (relative risk estimate 5.9; 95% confidence interval, 2.2, 16.1). The Ki-67 MoAb identifies a group of patients with rapidly fatal NHL for whom currently available chemotherapy is inadequate.

We describe a collection of 11 families with ≥ 2 generations of family members whose condition has been diagnosed as a hematologic malignancy. In 9 of these families there was a significant decrease in age at diagnosis in each subsequent generation (anticipation). The mean age at diagnosis in the first generation was 67.8 years, 57.1 years in the second, and 41.8 years in the third (P < .0002). This was confirmed in both direct parent-offspring pairs with a mean reduction of 19 years in the age at diagnosis (P = .0087) and when the analysis was repeated only including cases of mature B-cell neoplasm (P = .0007). We believe that these families provide further insight into the nature of the underlying genetic mechanism of predisposition in these families.

Background: Diagnosing multiple myeloma (MM) is challenging, because MM can present with a myriad of non-specific signs and symptoms, such as bone pain, fatigue, fractures, infection, within an elderly patient population (median age of MM diagnosis is 70 years). Patients with MM therefore experience significant delays in diagnosis, resulting in increased morbidity and reduced survival. In the primary care setting, the presence of bone pain in combination with laboratory abnormalities, such as hypercalcaemia or unexplained renal impairment, have a critical diagnostic role in MM. However, the occurrence of these clinical features prior to MM diagnosis within a population-based incident cohort of patients with MM has not been previously well described.

Adult T-cell leukemia (ATL) is a highly chemoresistant and usually fatal T-cell malignancy due to the human T-cell lymphotropic virus-1 (HTLV-1). After chemotherapy failure, antiretrovirals and interferon-α (IFN-α) produce brief responses followed by progression and death. More effective agents and new approaches to detect and treat minimal residual disease are needed. ATL cells express CD52, the target of the antibody alemtuzumab, which is active in a preclinical model of ATL and is cytotoxic for p53-deficient cells. A patient with refractory chronic ATL in transformation achieved longer than a 1-year complete hematologic response following 12 weeks of outpatient subcutaneous alemtuzumab. Persistent suppression of HTLV-1 viral load, even at recovery of T cells, after alemtuzumab and efficient in vitro complement-mediated cytotoxicity of primary ATL cells with mutated TP53 were observed. The unprecedented response and the profound suppression of HTLV-1 viral load observed in this patient suggest that further clinical investigation of alemtuzumab in ATL is warranted.

Introduction

No relevant conflicts of interest to declare.

Background: Acute traumatic coagulopathy occurs in both pediatric and adult trauma patients and is associated with an increased risk of mortality. Trauma patients not only have increased risk for hemorrhagic complications, but also are at increased risk for thrombosis due to multiple factors including local tissue injury, inflammation, and immobility. The complex underlying pathophysiology of coagulation abnormalities associated with traumatic injury have yet to be fully elucidated. Additionally, there are significant differences in the hemostatic system of pediatric patients compared to adults.

DNA methylation is one of the major epigenetic mechanisms that control T cell differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines, intermediates in active DNA demethylation. Here we demonstrate that TET2 regulates CD8+T cell differentiation in vivofollowing acute viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. However, following acute viral infection with LCMV-Armstrong, TET2 loss promotes early acquisition of a memory CD8+T cell fate in a cell-intrinsic manner without disrupting antigen-driven cell expansion or effector function. Integration of genome-wide methylation analysis and expression data suggest that TET2 loss leads to hypermethyation of the PRDM1genomic locus (encoding Blimp-1) and alters the relative expression of Blimp-1 and Bcl-6, two antagonistic transcriptional repressors known to direct CD8+T cell memory differentiation. Together, our data indicate that TET2 is an important regulator of CD8+T cell fate decisions.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

DNA methylation is one of the major epigenetic mechanisms that controls cellular differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases mediates active DNA demethylation through the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates. Here we demonstrate that TET2 regulates CD8+T cell differentiation in vivofollowing acute and chronic viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss enhances LCMV-specific CD8+T cell memory differentiation in a cell-intrinsic manner without disrupting antigen-specific cell expansion or cytokine production. However, TET2-deficient memory CD8+T cells exhibit altered recall responses with blunted re-expansion, retained expression of phenotypic memory markers and restricted re-expression of activation markers. During chronic viral infection with LCMV-clone 13, TET2 controls CD8+T cell expansion and alters differentiation. Importantly, though mice with T-cell specific loss of TET2 developed similar levels of CD8+T cell exhaustion as wild-type mice, TET2 loss specifically reduced PD-1 expression suggesting that TET2 may direct DNA demethylation of the PD-1 locus. Together, our data indicate that TET2 is an important regulator of CD8+T cells following both acute and chronic viral infections and suggest targeting epigenetic regulators have potential for enhancing antiviral immunity.

No relevant conflicts of interest to declare.

Nutritional support post allogeneic haematopoietic progenitor cell transplantation (HPCT) has proven efficacy in preventing malnutrition post-HPCT with resultant improved patient outcomes. However, there is no consensus as to the most efficacious approach to supplemental feeding post-allogeneic HPCT, leading to wide variation in clinical practice with respect to use of enteral (EN) vs.parenteral (PN) feeding, especially in adult units. We aimed to determine the tolerability and efficacy of EN vs.PN in adult patients undertaking allogeneic HPCT.

Nutritional support post allogeneic haematopoietic progenitor cell transplantation (HPCT) has proven efficacy in preventing malnutrition post-HPCT with resultant improved patient outcomes. However, there is no consensus as to the most efficacious approach to supplemental feeding post-allogeneic HPCT, leading to wide variation in clinical practice with respect to use of enteral (EN) vs. parenteral (PN) feeding, especially in adult units. We aimed to determine the tolerability and efficacy of EN vs.PN in adult patients undertaking allogeneic HPCT.Prospective randomized controlled study in adult patients >18yrs of age undertaking allogeneic HPCT with either myeloablative or reduced intensity conditioning (RIC). Informed consent was obtained before commencing conditioning, with patients randomized to receive either EN or PN after commencing HPCT conditioning if they could not maintain adequate oral nutritional intake, defined as maintaining >60% of daily caloric requirements orally. Patients with severe gastro-intestinal toxicity, including severe mucositis, were excluded from randomisation. The primary endpoint was tolerance of route of supplemental nutritional support, defined as <30% of patients needing to change to the alternative route of support for any reason.In total 38 patients were enrolled onto the study, including 9 patients (24%) undertaking myeloablative and 29 patients (76%) RIC HPCT. Only 19 patients (50%) required nutritional support post-HPCT. Of these 19 patients, only 9 (47%) were able to be randomized between EN (n=5) and PN (n=4), with 10 patients excluded from randomization due to presence of severe gastrointestinal toxicity in 7 (37%), markedly deranged liver function in 1 (5%), and withdrawal of consent in 2 (11%). The 5 patients randomized to EN met on average 74% of their goal nutrition and 100% required changing to PN due to (gastro-intestinal) intolerance. The 4 patients receiving PN met on average 91% of requirements, with none requiring change to EN.For adult patients undertaking myeloablative or RIC allogeneic HPCT, supplemental feeding with EN commencing at failure to maintain adequate oral nutritional intake is not feasible, due to the presence of significant gastrointestinal toxicity in these patients at this time. Further study is required to determine whether prophylactic nasogastric tube placement would improve the feasibility and tolerance of EN in this patient population.No relevant conflicts of interest to declare.

Saunthararajah: Cleveland Clinic Innovation: patent application for oral THU-decitabine., patent application for oral THU-decitabine. Patents & Royalties. Advani:Genzyme: Honoraria, Research Funding; Immunomedics: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Dabigatran is the first oral anticoagulant since warfarin to be approved by the FDA for prevention of stroke in patients with non-valvular atrial fibrillation. Unlike warfarin, dabigatran monitoring has not yet been recommended. However, dabigatran accounted for 3781 serious adverse events (including 542 deaths) as reported in QuarterWatch by the Institute for Safe Medicine Practices in 2011. This has prompted clinicians to consider situations in which drug level measurements may provide clinically useful information to guide dosage. Aside from managing the acutely bleeding patient, drug level monitoring may be useful in the outpatient setting. For example, establishing a baseline drug level may be relevant given the direct correlation with bleeding risk, especially in patients with decreased creatinine clearance. It can also assess patient compliance given the short half-life of the drug. We performed a retrospective analysis of patient samples that were tested for dabigatranlevels at our facility to determine whether drug levels were within the therapeutic range.Dabigatran levels were assayed for 172 frozen, citrated plasma samples from 127 patients (25 patients had multiple samples). The assay is a laboratory-developed, dilute thrombin clotting time test (CV ≤7.3%) that was validated by LC-MS/MS and is performed using drug-specific calibrators (Clin Chem 2012 abstract, in press). Specimens should be collected 90 to 120 minutes after the last dose and after ≥3 doses. The therapeutic range (64–443 ng/mL) was derived from the PETRO trial (van Ryn et al Throm Haemost2010). Patient demographics were correlated with drug levels, but clinical history was not available.Dabigatran levels were within the therapeutic range for 76 samples (44.2%), in the subtherapeutic range for 82 samples (47.7%), and in the supratherapeutic range for 14 (8.1%) samples. The median dabigatran level for the samples within the therapeutic range was 152 ng/mL. These samples also had a skewed distribution such that 44 (57.9%) were within the lowest quartile and 62 (81.6%) were within the lower half. Table 1 shows the distribution of dabigatranlevels by age and gender. Women ≥65 years of age were the most likely to have levels in the therapeutic range. Independent of gender, people aged <65 years were less likely to have levels in the therapeutic range. Four patients were not classified by age or gender.Of the 25 patients with multiple samples (2–6 samples/patient), 17 patients were consistently in or out of range: 8 therapeutic, 7 subtherapeutic, and 2 supratherapeutic. The remaining 8 patients were variable (Table 2).(1) A surprisingly high percentage of patients taking dabigatran were subtherapeutic. (2) The cause of the subtherapeutic levels is not readily discernable given the lack of clinical history, but may be explained by poor compliance, improper collection time, drug interactions, or decreased absorption (anecdotal evidence).No relevant conflicts of interest to declare.

In vitro expansion of UCB using cytokines has been pursued to overcome the limited stem cell content of a single UCB graft. We have previously demonstrated that a feeder layer of huMSC inhibits UCB HSC differentiation during short-term cytokine-driven expansion in vitro. The protein LIF has been shown to inhibit the differentiation of neurosphere stem cells during 3 week culture over a monolayer of murine stromal cells (C. Shih, et al., 2001). We sought to investigate the hypothesis that LIF secreted by bone marrow (BM) derived huMSCs is involved in inhibition of UCB HSC differentiation during short-term cytokine-driven expansion in vitro. BM derived huMSCs (third passage) were cultured at 2x10^6/ml in DMEM supplemented with 10% FBS. Supernatant was collected at 24, 36, 48, and 72 hours and analyzed for LIF secretion levels by ELISA (Quantikine). LIF secreted by huMSC was noted at all four time points, with peak secretion at 48 h (mean 52.1±3.3 pg/ml) (n=3). UCB was obtained according to institutional guidelines after normal full-term deliveries, collected into bags with citrate dextrose (Allegiance), and MNC were separated on a Histopaque-1077 (Sigma) density gradient. UCB CD133+ cells were isolated using AutoMACS magnetic cell sorter (Miltenyi) and surface stained for LIF receptor (LIF-R). Surface expression of LIF-R on gated CD133+ cells was 2.61%. Total LIF-R expression in isolated CD133+ cells was further confirmed by Western blot (n=3) using anti-LIF-R antibody (Chemicon). Isolated UCB CD133+ were plated in 24 well plates at 3.3x10^3/ml and cultured in StemPro™ media supplemented with 10% FBS, L-glutamine, penicillin, streptomycin and amphotericin B (Gibco). UCB CD133+ were culture-expanded for 96h with or without the addition of recombinant human LIF (Chemicon) (10ng/ml) in a combination of cytokines including: IL-3 (20 ng/mL), IL-6 (20 ng/mL), Flt-3L (100 ng/mL) (R&D), SCF (100 ng/mL), G-CSF (20 ng/mL), and EPO (0.1 U/mL) (Amgen Inc.). At 0, 48, and 96 h cell counts were obtained and flow analysis was performed including surface staining for: CD133, CD34, CD38, HLA-DR, CD33, CD71, and CD41B (Becton Dickinson). At 48 hours, higher cell counts in cultures without LIF were noted 6.3x10^4 ± 0.9/ml, compared with cultures with LIF 4.4x10^4 ± 0.8/ml. However, at 96 h cell counts equalized when comparing cultures with or without LIF at 7.510x10^4 ± 0.9/ml, and 7.8x10^4±0.9/ml respectively (n=6). Surface expression of differentiation markers on gated CD133+ cells did not differ when comparing cultures (n=3). In summary, we observed LIF secretion by MSC peaks at 48 h at a concentration 3 logs lower than that previously used to inhibit stem cell differentiation (10 ng/ml). Although LIF-R is expressed on CD133+ HSC, no difference in cell expansion nor surface phenotype of UCB 133+ cells was observed at early time points (96h) during expansion in cytokines with or without the addition of LIF. Taken together, huMSC mediated inhibition of cytokine-driven UCB HSC differentiation is not attributable to LIF secretion alone and may require direct cell contact between UCB CD133 HSC and huMSC. Studies are ongoing to determine whether LIF may augment huMSC-based UCB CD133 expansion.

Background. We demonstrated previously that intracoronary (IC) injection, in a dose-escalating fashion, of autologous bone marrow (BM)-derived mononuclear cells (MNCs) in normal pigs (n = 3) was safe. Although IC injection with the highest cell dose (15 x 10^6 cells) resulted in transient significantly reduced [Thrombolysis In Myocardial Infarction (TIMI) grade 1] flow in the injected artery, this did not result in myocardial infarction (MI), as determined by measurement of plasma biomarkers of myocardial necrosis and histopathologic examination. We further studied IC injection of autologous BM-derived MNCs in a porcine chronic myocardial ischemia model. Here we report the biodistribution of CM dioctadecyl tetramethylindocarbocyanine (CM-DiI)-labeled MNCs in solid organs and BM following IC injection. Methods. Chronic myocardial ischemia was induced in domestic pigs (n = 5) by surgical placement via lateral thoracotomy of an ameroid cuff around the distal left circumflex artery. Coronary angiography three weeks later confirmed absent or significantly reduced (TIMI grades 0 or 1, respectively) coronary blood flow. BM was aspirated (40 – 60 mL, mean 45.4 mL) from the right femur. MNCs were isolated by Ficoll density gradient centrifugation. Cells were labeled with CM-DiI at 37 °C for 20 minutes. A total of 15 x 106 MNCs in 5 mL of phosphate-buffered saline with autologous plasma was injected by IC delivery. Seven days later, BM was aspirated from the left femur, after which animals were euthanized. The heart and major organs were harvested for histological studies. Alternate sections were either stained with hematoxylin and eosin or left unstained for immunofluoroscence microscopy for CM-DiI visualization. Findings. Neither gross inspection nor histopathology revealed evidence of MI. Cell injection resulted in TIMI grade 1 coronary flow that resolved after 10 minutes and was not associated with vasospasm. There was no significant elevation in plasma biomarkers of myocardial necrosis at one or seven days following cell therapy. A limited number of labeled MNCs were observed in perivascular structures in myocardium at the injection site. Labeled cells were not seen elsewhere in either the myocardium or identified in lung, liver, or kidney. DiI-labeled MNC localization was observed in spleen harvested from one of the animals. BM aspirates, which did not allow for quantitation of cell numbers, indicated labeled MNCs. Conclusions. IC injection of DiI-labeled BM-derived MNCs resulted in cell localization in the perivascular areas of myocardium supplied by the injected vessel. Cell localization was not observed in most major solid organs, with spleen localization identified in only one animal. Labeled cells were identified in BM aspirates from the three animals injected with CM-DiI-labeled MNCs, suggesting a role for BM engraftment and repopulation as a possible mechanism for MNC localization in myocardium. It remains to be determined whether IC MNC injection resulted in initial homing to BM followed by migration to myocardium, or whether IC cell delivery resulted in initial myocardial deposition followed by BM localization. Ongoing studies include additional biodistribution studies of DiI-labeled BM- and umbilical cord blood-derived stem cell populations in a rat model of acute MI.

Recent reports have studied the use of cell populations from bone marrow, peripheral blood and umbilical cord blood (UCB) in mediating therapeutic angiogenesis in patients with coronary artery disease. We investigated the surface phenotype of UCB derived-mononuclear cells (MNC), and endothelial generating cells (EGC) by flow cytometry and in vitro functional migration studies. We used a murine hind-limb injury ischemia model to assess in vivo efficacy of the cell populations. METHODS: MNC were isolated by density centrifugation and CD133+ cells were isolated by magnetic separation (Miltenyi). EGC were derived by adherence of CD133− cells cultured 16h on fibronectin-coated tissue culture plates in EGM2 media (Clonetics). Cell characterization included surface phenotype determined by flow cytometry for monocyte markers CD14 and CD11b, stromal markers CD73 and CD105, KDR (VEGFR2), and the receptor for SDF-1, CXCR4. Transwell plates with 5μm collagen coated filters (Costar) were used to observe chemotactic migration of MNC or EGC towards SDF-1 (100ng/mL). Following a 3 hour incubation, the cells migrated to the bottom wells was counted by flow cytometry with TruCOUNT™ tubes (BD Biosciences. NOD/SCID mice underwent right femoral artery ligation and were injected with cytokines (EGM2 media, n=10), MNC (n=7) or EGC (n=8), 1X 106 cells/mouse. Laser Doppler blood flow measurements were recorded weekly for four weeks. RESULTS: Enhanced expression of CXCR4, CD105, KDR, CD14 and CD11b was found in the EGC cells generated after 16h culture on fibronectin. Surface Phenotype of UCB MNC and EGC CD14 CD11b CD73 CD105 KDR CXCR4 MNC (n≥5) 8.1 ± 2.7 22.7 ± 6.6 5.1 ± 2.2 6.7 ± 2.1 7.1 ± 2.1 28.5 ± 5.8 EGC (n=5) 67.7 ± 9.5 80.0 ± 4.8 7.1 ± 4.0 33.8 ± 5.8 37.6 ± 6.4 64.6 ± 7.8 Functional assays showed increased migration of both MNC and EGC to SDF-1 compared to control media (4.9 ± 2.9, n=2 and 3.2 ± 0.6, n=3 fold increases respectively). With VEGF as a chemoattractant MNC exhibited a 1.5 fold increase over the negative control (n=1) and EGC showed a 1.4 ± 0.3 fold increase (n=2). In the murine hind-limb ischemia model the ratio of ischemic/non-ischemic limb blood flow was used to compare vasculogenic potential. There was improvement of blood flow 14 days after injection of the EGC cells (p=0.019). On days 21 and 28, blood flow ratio was higher than control but was not statistically significant (p=0.06). The difference between MNC and EGC was not significant at any time point (p> 0.05). Histological studies are ongoing. CONCLUSION: In summary, UCB derived EGC exhibited monocyte and stromal surface antigen expression, migrated to an SDF-1 gradient, and mediated improved vascular blood flow. Ongoing studies are focused on direct cell vs. paracrine effects underlying observed neovasculogenesis mediated by EGC.

To assess in vitro angiogenesis, cellular co-culture assays have been utilized to study adherence, spreading, differentiation and proliferation, and migration of endothelial cells. Formation of tubule or capillary-like networks is influenced by the presence of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) but other factors provided by cell sources and/or direct contact with multiple cell types may facilitate this formation. The hypothesis of this study is that umbilical cord blood (UCB)-derived endothelial precursor cells (EPCs) may influence the formation of human umbilical vein endothelial cell (HUVEC) tubule structures during angiogenesis. Methods: UCB-derived EPCs were isolated from CD133negative cells after a 7-day culture on human fibronectin in EGM-2 media. Tubule formation was evaluated (passage 1–2, 20 x 103 or 2 x 103 cells) by adding HUVECs without or with EPCs to cultures of human bone marrow-derived mesenchymal stromal cells (MSCs) under normoxic (20%) conditions (37°C, 5% CO2, containing VEGF, epidermal growth factor, FGF, insulin-like growth factor, heparin, hydrocortisone, and ascorbic acid in EGM-2 medium) for a 2-week period. HUVECs were added to cultures without or with labeling with Vybrant® CM-DiI which allows the temporal observation of tubule formation progress and cellular incorporation. Final tubule formation was confirmed using a primary anti-CD31 (PECAM) antibody followed by a FITC-conjugated secondary antibody for signal amplification. Results: After 2–4 days, linear aggregates of labeled HUVECs (2-D arrangement) were observed. After 14 days, there was remodeling of HUVECs into the development of a 3D network of linear and branched tubule structures. EPCs facilitated the formation of tubules affecting both the extent of tubule formation and also enhanced proliferation of HUVEC cells. A minority (< 5%) of EPCs were incorporated into developing tubules (estimated using CM-Dil-labeled EPCs). To quantify tubule formation, digital pictures of representative areas of culture wells (2–4/well) were acquired. Using Image Pro Plus software, tubules were quantified using multi-parameter analysis with respect to length, area, and perimeter. The presence of EPCs (equal to the number of added HUVECs) significantly enhanced all parameters. In comparison to control samples, the presence of EPCs increased the area, perimeter and size by 15.2-fold, 3.4-fold, and 3.2-fold, respectively. Confocal microscopy revealed that the co-cultures formed anatamoses, indicating the formation of a connected network. Conclusions: Taken together, these results suggest that the presence of cord blood-derived EPCs facilitate tubule formation and development via a heterotypic cell-cell interaction without integrating into the angiogenic structures. Further studies will evaluate the secretion of cytokines and growth factors.