Search

Pre-existing central nervous system (CNS) involvement may influence referral for autologous haematopoietic cell transplantation (AHCT) for patients with non-Hodgkin lymphoma (NHL). The outcomes of 151 adult patients with NHL with prior secondary CNS involvement (CNS(+) ) receiving an AHCT were compared to 4688 patients without prior CNS lymphoma (CNS(-) ). There were significant baseline differences between the cohorts. CNS(+) patients were more likely to be younger, have lower performance scores, higher age-adjusted international prognostic index scores, more advanced disease stage at diagnosis, more aggressive histology, more sites of extranodal disease, and a shorter interval between diagnosis and AHCT. However, no statistically significant differences were identified between the two groups by analysis of progression-free survival (PFS) and overall survival (OS) at 5 years. A matched pair comparison of the CNS(+) group with a subset of CNS(-) patients matched on propensity score also showed no differences in outcomes. Patients with active CNS lymphoma at the time of AHCT (n = 55) had a higher relapse rate and diminished PFS and OS compared with patients whose CNS lymphoma was in remission (n = 96) at the time of AHCT. CNS(+) patients can achieve excellent long-term outcomes with AHCT. Active CNS lymphoma at transplant confers a worse prognosis.

Background:The primary objective was to describe infectious complications in children with acute myeloid leukemia from presentation to the healthcare system to initiation of chemotherapy and to describe how these infections differ depending on neutropenia.Methods:We conducted a retrospective, population-based cohort study that included children and adolescents with acute myeloid leukemia diagnosed and treated at 15 Canadian centers. We evaluated infections that occurred between presentation to the healthcare system (for symptoms that led to the diagnosis of acute myeloid leukemia) until initiation of chemotherapy.Results:Among 328 children, 92 (28.0%) were neutropenic at presentation. Eleven (3.4%) had sterile-site microbiologically documented infection and four had bacteremia (only one Gram negative). Infection rate was not influenced by neutropenia. No child died from an infectious cause prior to chemotherapy initiation.Conclusion:It may be reasonable to withhold empiric antibiotics in febrile non-neutropenic children with newly diagnosed acute myeloid leukemia until initiation of chemotherapy as long as they appear well without a clinical focus of infection. Future work could examine biomarkers or a clinical score to identify children presenting with leukemia and fever who are more likely to have an invasive infection. © 2013 Portwine et al.

From 1 November 1992 through 1 May 1993 and from 1 November 1993 through 1 May 1994, we conducted a prospective surveillance study at the University of Texas M.D. Anderson Cancer Center (Houston) to evaluate the role of community respiratory virus infections in hospitalized adult bone marrow transplant (BMT) recipients, Respiratory secretions were obtained from all adult BMT recipients with acute respiratory illnesses. During these two winters, a community respiratory virus was isolated from 37 (36%) of 102 patients and 30 (26%) of 115 patients, respectively. Approximately half (49%) of these infections were due to respiratory syncytial virus (RSV); the remainder were due to influenza virus (18%), picornaviruses (18%), parainfluenza virus (9%), or adenovirus (6%). Fifty-eight percent of these infections were complicated by pneumonia, with an associated mortality of 51%. The pneumonias that complicated RSV infection were almost exclusively viral in origin and were associated with a mortality of 100% if not treated promptly with antiviral agents. In contrast, many of the pneumonias that complicated the other viral infections, such as influenza, appeared to be either self-limited viral pneumonias or secondary bacterial or fungal pneumonias. Community respiratory viruses are frequent causes of acute respiratory illnesses in adult BMT recipients hospitalized during the winter and are associated with substantial morbidity and mortality.

Background. Infection continues to be a major problem for children with acute myeloid leukemia (AML). Objectives were to identify factors associated with infection, sepsis, and infectious deaths in children with newly diagnosed AML.Methods. We conducted a retrospective, population-based cohort study that included children ≤18 years of age with de novo, non-M3 AML diagnosed between January 1995 and December 2004, treated at 15 Canadian centers. Patients were monitored for infection from initiation of AML treatment until recovery from the last cycle of chemotherapy, conditioning for hematopoietic stem cell transplantation, relapse, persistent disease, or death (whichever occurred first). Consistent trained research associates abstracted all information from each site.Results. 341 patients were included. Median age was 7.1 years (interquartile range [IQR], 2.0-13.5) and 29 (8.5%) had Down syndrome. In sum, 26 (7.6%) experienced death as a first event. There were 1277 courses of chemotherapy administered in which sterile site microbiologically documented infection occurred in 313 courses (24.5%). Sepsis and infectious death occurred in 97 (7.6%) and 16 (1.3%) courses, respectively. The median days of corticosteroid administration was 2 per course (IQR, 0-6). In multiple regression analysis, duration of corticosteroid exposure was significantly associated with more microbiologically documented sterile site infection, bacteremia, fungal infection, and sepsis. The only factor significantly associated with infectious death was days of corticosteroid exposure (odds ratio, 1.05; 95% confidence interval, 1.02-1.08; P =. 001).Conclusions. In pediatric AML, infection, sepsis, and infectious death were associated with duration of corticosteroid exposure. Corticosteroids should be avoided when possible for this population. © The Author 2012. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.

Respiratory syncytial virus (RSV) has been demonstrated to be an important cause of life-threatening pneumonia in adult bone marrow transplant recipients; however, its role in other immunocompromised adults has not been defined. We prospectively studied all adult patients with leukemia who were hospitalized at M. D. Anderson Cancer Center (Houston) during a 1-year period (November 1993 through October 1994). During a 19-week period when RSV was prevalent in the community, it was isolated from 9 (10%) of 87 patients with leukemia who developed an acute respiratory illness. In 6 (75%) of 8 patients with profound chemotherapy-induced myelosuppression, the RSV infection was complicated by pneumonia, with an 83% mortality rate. RSV appears to be an important cause of severe and often fatal pneumonia in myelosuppressed patients with leukemia.

Summary Radiofrequency endometrial ablation is an experimental technique which requires a minimum of training and appears to be safe so long as a strict protocol is maintained. The success rate in over 1000 patients in multiple centes gives an incidence of amenorrhoea or oligomenorrhoea in excess of 80%. Therefore, as long as patients are strictly selected more than eight out of 10 hysterectomies for dysfunctional uterine bleeding can be avoided. The technique is particularly suitable for women with severe cardiopulmonary disease because there is no risk of pulmonary or cerebral oedema due to fluid intravasation.

We evaluated single nucleotide polymorphisms (SNPs) associated with infection risk in children with newly diagnosed acute myeloid leukaemia (AML). We conducted a multicentre, prospective cohort study that included children aged ≤18 years with de novo AML. DNA was isolated from blood lymphocytes or buccal swabs, and candidate gene SNP analysis was conducted. Primary outcome was the occurrence of microbiologically documented sterile site infection during chemotherapy. Secondary outcomes were Gram-positive and -negative infections, viridans group streptococcal infection and proven/probable invasive fungal infection. Interpretation was guided by consistency in risk alleles and microbiologic agent with previous literature. Over the study period 254 children and adolescents with AML were enrolled. Overall, 190 (74.8%) had at least one sterile site microbiologically documented infection. Among the 172 with inferred European ancestry and DNA available, nine significant associations were observed; two were consistent with previous literature. Allele A at IL1B (rs16944) was associated with decreased microbiologically documented infection, and allele G at IL10 (rs1800896) was associated with increased risk of Gram-positive infection. We identified SNPs associated with infection risk in paediatric AML. Genotype may provide insight into mechanisms of infection risk that could be used for supportive-care novel treatments.

The aim of this study was to investigate the effects of high-dose corticosteroids on behavior and quality of life of children with acute lymphoblastic leukemia (ALL) in maintenance therapy. Forty-three families (patient and/or parent) completed the Child Behavior Checklist (CBCL), the Pediatric Quality of Life Inventory (PedsQL) Cancer module questionnaires, a checklist of common complaints, and a descriptive intervention data questionnaire, on and off steroids, each child serving as his/her own control. Children 5 years and younger had higher CBCL scores in the somatic complaints, affective problems, internalizing, externalizing, and total problem scales, when comparing "on-steroid" to "off-steroid" periods (P

Cytogenetics play a major role in determining the prognosis of patients with acute myelogenous leukemia (AML). However, existing cytogenetics classifications were developed in chemotherapy-treated patients and might not be optimal for patients undergoing allogeneic hematopoietic cell transplantation (HCT). We studied 821 adult patients reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) who underwent HCT for AML in first or second complete remission between 1999 and 2004. We compared the ability of the 6 existing classifications to stratify patients by overall survival. We then defined a new scheme specifically applicable to patients undergoing HCT using this patient cohort. Under this scheme, inv(16) is favorable, a complex karyotype (4 or more abnormalities) is adverse, and all other classified abnormalities are intermediate in predicting survival after HCT (5-year overall survival, 64%, 18%, and 50%, respectively; P = .0001). This scheme stratifies patients into 3 groups with similar nonrelapse mortality, but significantly different incidences of relapse, overall and leukemia-free survival. It applies to patients regardless of disease status (first or second complete remission), donor type (matched related or unrelated), or conditioning intensity (myeloablative or reduced intensity). This transplantation-specific classification could be adopted for prognostication purposes and to stratify patients with AML and karyotypic abnormalities entering HCT clinical trials. American Society for Blood and Marrow Transplantation.

BACKGROUND: Recipientsof allogeneic HCT remain vulnerable to a heightened risk of reactivation of otherwise latent viral infections owing to a compromised immune system early after transplantation. Uncontrolled reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disorder (PTLD) is one of such major complications after T-cell depleted HCT. Recovering within weeks after transplantation and being first in line of defense against viral infections, natural killer (NK) cells are deemed important in the immunopathogenesis of EBV complications. Their role however remains elusive. NK cell responses are regulated by a series of activating and inhibitory cell surface receptors, central to which are the Killer Immunoglobulin-like Receptors (KIR). Through these receptors NK cells discriminate healthy cells from 'altered' self-cells by scaling the perturbations in HLA expression after viral transformation of the target cell. Here, we set out to determine whether and how KIR gene and motifs' content of HCT donors and/or recipients influences the development of PTLD after allo-HCT. STUDY DESIGN: Hypothesizing that diverse NK cell receptor repertoires can titrate NK cell functional responses to EBV infections/reactivation and can potentially modify the risk of developing PTLD, we determined the KIR gene repertoires of 356 HLA-matched donor-recipient pairs of first allo-HCT and 50 healthy donors through Next Generation Sequencing of the KIR locus on the Illumina MiSeq platform. Based on the presence/absence and number of copies of individual genes, the KIR genotypes were determined and classified into four common centromeric (cA01, cB01, cB02 and cB03) and two telomeric (tA01 and tB01) motifs along with their variants. PBMNCs from KIR typed healthy volunteers were stimulated with EBV-transformed target cells to enumerate NK cell response to EBV (degranulation and/or IFNγ production) as a function of KIR gene content and motifs' distribution using a multicolor flow cytometry-based assay. Effect of KIR gene profile on development of PTLD was analyzed using binomial competing risks regression statistics. Distribution of NK cell functional response across various KIR characterized groups was analyzed using Mann-Whitney U statistics. RESULTS: Donor telomeric A motifs (tA01, KIR3DL1+ve KIR2DS4+ve; KIR3DS1/2DS1+/-ve), strongly protected against PTLD (p=0.0001, SHR=0.17; Figure 1). An increased protection against PTLD with increasing number of tA01 was noted with at least one copy required for a significant protective effect (Figure 1B). Copy number analysis of tA01 gene contents yielded similar associations. Further, the number of EBV induced functional NK cell subsets were significantly higher in individuals with than without KIR genotypes containing tA01 motifs (Figure 2 A-C) and was found to be increasing with an increasing number of tA01 copies (Figure 2 A'-C'). There was no influence of recipients' KIR repertoire on the risk of developing PTLD CONCLUSIONS: NK cell responsiveness, a function of KIR gene repertoire has a profound effect on the development of PTLD. Appropriately characterized KIR gene profile based identification of HCT recipients at high risk of developing PTLD will enable closer monitoring of EBV DNAemia and facilitate prompt therapy. Figure 1. Donor KIR telomeric A motif (tA01) protects against the risk of developing PTLD (A). Presence of at least one copy of donor KIR tA01 motif confers significant protection from PTLD (B) Figure 1. Donor KIR telomeric A motif (tA01) protects against the risk of developing PTLD (A). Presence of at least one copy of donor KIR tA01 motif confers significant protection from PTLD (B) Figure 2. KIR telomeric A motifs (tA01) titrate NK cells' functional response to Epstein-Barr virus infected cells (A-C), with and increasing %functional NK cells and subsets (measures as expressing CD107a, IFN-γ, or both) are observed with increasing tA01 motifs' copies (A'-C') Figure 2. KIR telomeric A motifs (tA01) titrate NK cells' functional response to Epstein-Barr virus infected cells (A-C), with and increasing %functional NK cells and subsets (measures as expressing CD107a, IFN-γ, or both) are observed with increasing tA01 motifs' copies (A'-C') Disclosures No relevant conflicts of interest to declare.

Although reduced intensity (RIC) and nonmyeloablative (NMA) conditioning regimens have been used for over a decade, their relative efficacy versus myeloablative (MA) approaches to allogeneic hematopoietic cell transplantation (HCT) in patients with acute myelogenous leukemia (AML) and myelodysplasia (MDS) is unknown. We compared disease status, donor, graft and recipient characteristics with outcomes of 3731 MA with 1448 RIC/NMA procedures performed at 217 centers between 1997 and 2004. Five year univariate probabilities and multivariate relative risk (RR) outcomes of relapse, transplant related mortality (TRM), disease free survival (DFS) and overall survival (OS) are reported. Adjusted OS at 5 years was 34%, 33%, and 26% for MA, RIC and NMA transplants, respectively. NMA conditioning resulted in inferior DFS and OS but there was no difference in DFS and OS between RIC and MA regimens. Late TRM negates early decreases in toxicity with RIC and NMA regimens. Our data suggest higher regimen intensity may contribute to optimal survival in patients with AML/MDS, suggesting roles for both regimen intensity and graft vs. leukemia in these diseases. Prospective studies comparing regimens are needed to confirm this finding and determine the optimal approach to patients who are eligible for either MA or RIC/NMA conditioning.

Pediatric bone marrow transplantation (BMT) for various diseases can lead to endocrine system dysfunction owing to preparative regimens involving chemotherapy and radiation therapy. We assessed the prevalence of post-BMT endocrine complications in children treated at the Alberta Children's Hospital (ACH) from 1991 to 2001. Time of onset of endocrine dysfunction, underlying disease processes, chemotherapy, radiation therapy and age at BMT were characterized. Subjects of

Interference with the molecular mechanisms that generate tumor supportive niches in the bone microenvironment is a rational approach to inhibit the growth of hematological malignancies. However, the advancement of knowledge in this area has been slowed down by the lack of in vitro models to facilitate the screening of potential candidate agents. The rare cases of acute lymphoblastic leukemia (ALL) in children presenting with extensive bone involvement may represent an exaggerated form of some aspects of the normal tumor-bone interactions. Thus, these cases can provide insight into processes that are otherwise challenging to uncover. The authors describe the case of a 6-year-old child who presented with severe osteopenia that resolved at the time of leukemic remission. Compared to control sera, serum taken at disease presentation contained increased levels of a group of osteolytic cytokines and was effective in activating preosteoclast cells in culture. Based on these findings, the authors describe an experimental model to identify agents that would interfere with leukemia mediated osteolytic process.

ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 to induce apoptosis in malignant cells and has shown promise as an effective anti-leukemic agent in pediatric preclinical tests. This study focuses on the effects of ABT-737 on leukemia cells with MLL rearrangement and identifies some of the biological correlates of its activity.Cells were cultured in the presence of increasing concentrations of ABT-737 alone or in combination with other agents. After 4 days in culture, cell growth inhibition was measured by Alamar blue assay. The expression and activation of potential intracellular targets of ABT-737 activity were determined by Western blot analysis.Significant Bcl-2 expression was detected in all infant leukemia cells investigated. ABT-737 induced cell death in all cell lines studied although the IC(50) values differed somewhat between cell lines. Western blot analysis identified the effects of ABT-737 on survival and apoptosis-regulatory proteins PARP, caspase-8, and cytochrome-c. Drug combination studies indicated synergy with distinct anti-neoplastic agents, including the multi-tyrosine kinase inhibitor sunitinib. This effective drug synergy appears to be mediated by the combined inhibition of Bcl-2 and intracellular signaling pathways.We describe the in vitro studies to demonstrate the activity and drug combinability of ABT-737 against MLL rearranged leukemia cells. In addition, identification of the molecular changes that occur in the presence of ABT-737 provides information regarding effective target validation and target modulation analyses in future clinical trials.

In children with acute lymphoblastic leukemia (ALL) with isolated central nervous system (CNS) relapse and a human leucocyte antigen (HLA)-matched sibling, the optimal treatment after attaining second remission is unknown. We compared outcomes in 149 patients enrolled on chemotherapy trials and 60 HLA-matched sibling transplants, treated in 1990-2000. All patients achieved a second complete remission. Groups were similar, except the chemotherapy recipients were younger at diagnosis, less likely to have T-cell ALL and had longer duration (> or = 18 months) first remission. To adjust for time-to-transplant bias, left-truncated Cox's regression models were constructed. Relapse rates were similar after chemotherapy and transplantation. In both treatment groups, relapse rates were higher in older children (11-17 years; RR 2.81, P=0.002) and shorter first remission (< 18 months; RR 3.89, P

Busulfan (BU) dose adjustment following therapeutic drug concentration monitoring improves outcomes of hematopoietic stem cell transplantation (HSCT). Further improvement could be achieved through genotype-based BU dose adjustments. Studies with large and homogeneous samples are essential to establish the utility of genetic factors as a predictor of BU dose and outcomes of HSCT. In this multicentric study, we included 139 children aged between 0.1 to 19.9 years (65 females) receiving BU based myeloablative conditioning regimen prior to allogeneic HSCT. Genetic predictors of BU plasma levels, dose adjustment, and clinical outcomes post HSCT were prospectively evaluated. Patients received BU in a conventional 6 h dosing schedule as a two hours infusion for four days in combination with cyclophosphamide (CY; n=106), or CY and etoposide (15) or melphalan and CY (15).The remaining patients received BU with melphalan (2) or fludarabine (1). BU first dose pharmacokinetic parameters (PKs) in patients were estimated and were adjusted to steady state concentrations (Css) of 600-900 ng/mL. All the patients were genotyped for defining GSTA1*A1,*A2, *B1 and *B2 haplotypes, GSTM1, GSTT1 null alleles, CYP2C9*2,*3, CYP2C19*2,*17, CYP2B6*5, *9 and GSTP1 1578A>G, GSTP1 2293 C>T polymorphisms. Standard criteria were followed for defining clinical outcomes of HSCT such as neutrophil recovery, platelet recovery, acute graft versus host disease (aGvHD), sinusoidal obstruction syndrome (SOS), event-free survival (EFS), non-relapse mortality (NRM), hemorrhagic cystitis (HC), and graft failure (Ther Drug Monit. 2014;36:93-9; Bone Marrow Transplant. 2013; 48:939-46; Pharmacogenomics J. 2014; 14:263-71). PKs such as BU clearance (CL), area under the curve concentration (AUC), and Css were compared among genotype groups using non-parametric tests. The PKs and genetic variants or haplotypes were also associated with the clinical outcomes individually or in combinations in both univariate and multivariate analysis. Statistical significance was set at p Disclosures No relevant conflicts of interest to declare.

Introduction: High resolution human leukocyte antigen (HLA) matching is regarded as a prerequisite for the clinical success of allogeneic haematopoietic cell transplantation (HCT). However, studies have reported immunogenetic factors other than HLA also plays an important role in preventing and controlling post HCT complications, in particular graft-versus-host disease (GVHD) and infections. Cytokines and cytokine receptors, which are the key regulators of the immune surveillance against infections and/or allo-immune responses, are important candidates. The genetic control of cytokine production is evidenced by polymorphisms in cytokine gene regulatory regions resulting in low, moderate, or high cytokine production. Here, we evaluated the prognostic significance of cytokine genetic variants known to impact cytokine production on different outcomes of allogeneic HCT. Materials and Methods: A total of 793 subjects were accrued, including 360 adult allo-HCT donors (discovery cohort; n=240, and validation cohort; n=120), 382 HCT allo- recipients and 50 healthy individuals. All subjects of the discovery cohort and healthy individuals were analyzed for 22 single nucleotide variants located in the regulatory and/or exonic regions of 13 cytokine or cytokine receptor gene using sequence-specific primer based assay. All subjects of the validation cohort were genotyped SNPs located in the IL10 and IL1R gene promoter region by direct sequencing. . CMV specific immune response was assessed by stimulating PBMNCs from healthy individuals with CMV lysate and CMV peptide-pp65 followed by enumeration of IFN-g producing and CD107a expressing (degranulating) MNCs, T cells and their subsets. Results: Cytokine gene variants of donors and not that of recipients appear to influence post HCT complications. Two of these variant system- SNPs leading to low production of IL10 and high production of IL1R showed strong correlation with GVHD and posttransplant CMV infections respectively. Allo-HCT recipients who have received graft from donors carrying low IL-10 producing gene variants (AA) at IL-10 -1082G/A and (ATA/ATA at IL-10 -1082G/A, -819C/T and -592C/A) have high incidence of significant GVHD (defined as grade II-IV acute GVHD and/or chronic GVHD requiring systemic immunotherapy) (P=0.008, HR= 2.8, Figure 1; and P=0.03, HR= 1.8 respectively) compared to recipients who have received graft from donors carrying high producer IL-10 gene variants. Allo-HCT recipients who have received graft from donors carrying low IL-1R producing genotype (-1970 CC) showed high incidence of CMV reactivation (p=0.01; HR=2.1, Figure 2) in comparison to the HCT recipients receiving grafts from donors carrying high IL1-R producing genotype (-1970 GG). Further, the in-vitro culture analysis in healthy individuals showed that IL1R+ cells have 3-5 fold stronger anti-CMV immune responses (IFNg+ and/or CD107a+ cells) in comparison to IL1R- cells. Conclusions: Genetic predisposition to low IL-10 production is a strong predictor of GVHD while that tohigh production of IL-1R confers strong protection against CMV reactivation after allogeneic HCT. Thefindings implicate the importance of prospective assessment of cytokine gene variants to improve allogeneic HCT donor selection. Figure 1: Figure 1:. High incidence of significant GVHD in HCT recipients receiving grafts from donors carrying low IL-10 producing genotypes (-1082 AA) than donors carrying high IL-10 producing genotype (-1082 GG). Figure 2: Figure 2:. High incidence of CMV reactivation in HCT recipients receiving grafts from donors carrying low IL-1R producing genotype (-1970 CC) than donors carrying high IL-1R producing genotype Disclosures No relevant conflicts of interest to declare.

Aim An immunocompromised state after hematopoietic cell transplantation (HCT) leads to a deficient anti-cytomegalovirus (CMV) immune response. This often culminates into episodes of posttransplant CMV reactivation and complications. Cytokines and their receptors act as chief mediators of anti-viral immune response. Genetic control of cytokine production is evidenced by polymorphisms in cytokine gene regulatory regions resulting in low, moderate, or high cytokine production profiles. In the present investigation, we assessed whether gene variants of cytokine or their receptors influences post HCT CMV complications and anti-CMV immune response. Methods A total of 240 allogeneic HCT donors and 50 healthy individuals were genotyped for 22 single nucleotide variants located in the regulatory and/or exonic regions of 13 cytokine or cytokine receptor genes. CMV specific immune response was assessed by stimulating PBMNCs from healthy individuals with CMV lysate and CMV peptide-pp65 followed by enumeration of IFN-γ producing and CD107a expressing (degranulating) MNCs, T cells and their subsets. Results Allogeneic HCT recipients receiving grafts from donors carrying low IL-1R producing genotype (-1970 CC) had high incidence of CMV reactivation (p = 0.01; Hazard ratio = 2.1, Fig. 1 ) in comparison to the HCT recipients receiving grafts from donors carrying high IL1-R producing genotype (-1970 TT). Further, the analysis in healthy individuals showed that IL1R+ cells have 3-5-fold stronger anti-CMV immune response (IFNγ+ and/or CD107a+ cells) in comparison to IL1R- cells. Conclusions High IL-1R producing gene variants confer strong protection against CMV reactivation after allogeneic HCT. The conclusions are further supported by the finding that anti-CMV immune response is dominated by IL1R+ MNCs.

Background Often referred to as the ‘third lymphocytes’, Natural Killer (NK) cells are critical to early control of viral infections and immune-surveillance of tumors and leukemias. Regulation of NK cell responses is mediated by one or more HLA class-I specific inhibitory receptors, (chiefly, of the Killer immunoglobulin-like receptor (KIR) family), in a way that it can only be triggered when the inhibitory signals are either diminished or are overridden by activating signals. Given the remarkable genetic diversity demonstrated by the KIR gene cluster, it is hypothesized that different KIR gene repertoires characterized by differences in activating and inhibitory gene content could be associated with differential capacities of NK cells (and certain T cell subsets) to eliminate leukemic target cells. Methods A total of 218 subjects including healthy individuals ( n = 124) and patients of myelogenous ( n = 62) and lymphocytic ( n = 32) leukemia respectively were analyzed for KIR gene repertoire distribution using Luminex™ based assay. Differences in average incidences of individual KIR genes and haplotypes among all three groups were tested for significance. Results Significantly reduced incidences of activating KIR2DS3 ( p = 0.009; OR = 5.7) and KIR3DS1 ( p = 0.01; OR = 3.1) were observed in patients suffering from lymphoid malignancies and not in patients suffering from myeloid malignancies. KIR gene repertoires containing ⩽ 1 activating KIR were significantly more often represented by the lymphocytic leukemia patients ( p = 0.03; OR = 3.5) as compared to controls who most often presented ⩾5 activating KIR. Conclusions This finding supports the hypothesis that an absence of appropriate activation signals may provide lymphocytic leukemias an escape mechanism from immune surveillance by NK cells.

Background Complications of allogeneic hematopoietic cell transplantation (HCT), primarily graft versus host disease (GVHD) and relapse of the underlying disease are substantial. In spite of high resolution HLA matching incidences of clinically significant GVHD and disease relapse among Albertan HCT recipients remain as high as 35% and 20%, respectively. Unfortunately, strategies/treatments aiming to control GVHD in most cases lead to increase in the rate of disease relapse and infections. In the recent years, the natural killer (NK) cell genetic system, regulated by the activating and inhibitory Killer Immunoglobulin-like Receptors (KIR) has garnered substantial research interest as a modifier of HCT outcomes. Here we set out to determine the influence of KIR matching between HCT donor and recipient pairs on allogeneic HCT complications. Study design KIR gene repertoires of 200 allo-HCT pairs were obtained by a Luminex-based rSSO method. Donor (D) and recipients (R) were ‘matched’ for KIR genotypes when both carried either AA (two copies of group A haplotypes) or B/x (at least one copy of group B haplotype) genotypes. Effect of KIR matching on significant GVHD (described as grade 2–4 acute GVHD or chronic GVHD needing systemic therapy) as well as disease relapse was analyzed using competing-risks regression and Cox proportional hazard statistics. Results A significant protection against GVHD was conferred upon when both the donor and recipient were matched for the KIR genotypes (HR = 1.7; p = 0.03) without any effect on disease relapse (HR = 1.2; p = 0.57). Recipients of a KIR genotype matched graft appeared to have an improved survival. Conclusions Since GVHD is concomitant with graft-vs-leukemia (GVL) reaction, strategies to decrease GVHD in most cases have resulted in increased relapse. Matching donor and recipients for KIR genotypes can offer a significant protection against GVHD without increasing the risk of disease relapse.

Soft tissue and bone sarcomas of the extremities can be difficult to eradicate, and standard treatment may require limb amputation. New therapies to decrease tumor size could improve the effectiveness of treatment and decrease the frequency of limb amputation. Cytosine deaminase (CD)-based gene therapy has been shown to be effective in decreasing growth of solid tumors when animals with CD-expressing tumor cells are treated with 5 fluorocytosine (5FC), an inert prodrug that is converted to 5-fluorouracil (5FU) by CD. In this investigation, we used a novel CD-containing fusion gene to determine whether CD-based gene therapy affected soft tissue or bone sarcomas. The novel fusion gene (NGFR-CD) encodes for a protein with extracellular and transmembrane domains of human nerve growth factor receptor (NGFR) and cytoplasmic CD. Murine 2472 (2) sarcoma cells were transduced with fusion genes containing either the bacterial (NGFR-(b)CD) or yeast (NGFR-(y)CD) CD gene. 5FC treatment killed NGFR-(b)CD- and NGFR-(y)CD-transduced sarcoma cells in vitro through direct and bystander effects (P0.01). In contrast, 5FC treatment of mice with s.c. 2NGFR-(b)CD or 2NGFR-(y)CD tumors affected only 2NGFR-(y)CD tumors. 5FC had no effect on growth of NGFR-(b)CD tumors but caused significant decrease in the size of 2NGFR-(y)CD tumors (51 +/- 60 versus 938 +/- 767 mm(3), treated versus control, P0.01). Evaluation of bystander killing in vivo revealed significant tumor killing, with a 5-fold reduction in s.c. tumor volume evident in saline versus 5FC-treated mice when tumors were comprised of 90% 2472 cells and 10% 2NGFR-(y)CD selected for fluorescence-activated cell sorting (P0.01). Bone sarcomas were eliminated in 9 of 10 5FC-treated mice, compared with 11.8 +/- 6.0 mm(2) in saline-treated mice (P0.002). In addition, 5FC treatment of bone sarcomas caused a significant reduction in cancer-induced bone destruction (P0.002) and resulted in a reduction in the number of osteoclasts. Finally, 5FC treatment had no effect on animal weight or survival, whereas doses of 5FU providing equivalent tumor reduction as 5FC resulted in treatment-associated deaths and significant weight loss (P0.001).

Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.

Introduction Despite the considerable progress that has been made in past two decades, relapsed or refractory leukemia still remains a leading cause of death in children. Acute and long term toxicities prevent significant further intensification of current chemotherapeutic regimens. Hence, effective pre-clinical data on new agents and novel therapeutic approaches are urgently needed. Recent studies have established proteasome inhibition as a distinctive and effective way to induce cytotoxicity in tumor cells that have acquired resistance to conventional chemotherapy. Although they represent a major advancement in the treatment of various hematological malignancies, the first generation proteasome inhibitors exhibit measurable off-target toxicities and the eventual development of resistance. Carfilzomib (CFZ) is a selective proteasome inhibitor that is structurally distinct from bortezomib and has shown efficacy and favorable toxicity profile in multiple myeloma patients. Mechanistically, carfilzomib has been shown to irreversibly bind and inhibit the chymotrypsin-like activity of the 20S proteasome and to cause the accumulation of polyubiquinated proteins resulting in cell cycle arrest, apoptosis, and suppression of tumor growth. Methods A diverse panel of pediatric leukemia derived cell lines and primary leukemia specimens (n = 12) were used to evaluate CFZ induced in vitro cytotoxicity using the Alamar blue assay. These cell lines include leukemia with abnormal FLT3 (FLT3-ITD and over-expression), Bcr-Abl fusion, extremely high white blood cell count variants, mixed lineage leukemia and Juvenile Myelomonocytic Leukemia (JMML) cells that are hyper-stimulated with exogenous GM-CSF. A luminescent based technique that individually measures protease activities associated with the proteasome complex in cultured cells (Promega, Cell-Based Proteasome-Glo Assay) was used to evaluate the mechanism of CFZ activity in these cells. Drug combination studies were carried out with etoposide, cytarabine, sorafenib and mefloquine using Chou and Talalay methodology. Target modulation, induction of apoptosis and the modulation of autophagy were evaluated by Western Blot analysis of cells treated with CFZ at defined time periods. Results Carfilzomib induced effective cytotoxicity in all leukemia cells tested (IC50 mean = 7 nM, range = 0.2 – 10 nM). Cell based proteasome assays confirmed the targeted and specific activity of CFZ in these cells. Infant leukemia cells with FLT3 over-expression were highly sensitive to CFZ followed by cells with ITD. Primary JMML cells that showed high growth stimulation with GM-CSF were also significantly affected by CFZ (IC50 = 0.2 nM). Although the extent of drug synergy varied between AML and ALL cells, CFZ synergized with all four agents (Combination Index (CI) mean = 0.47, range = 0.2 – 0.9). Induction of apoptosis by CFZ was evidenced by the increase in the active fragments of caspase 7 and 8 and PARP cleavage. CFZ also modulated autophagy by showing concentration regulated changes in p62 and LC3B. However, this effect appears to be restricted to AML cells. In vitro clonogenic assays using normal human CD34+ cells showed that even at 10 nM concentration, CFZ has no detectable inhibition on erythroid or myeloid colony formation. Discussion Carfilzomib is a potent, selective and irreversible inhibitor of the ubiquitin-proteasome pathway in cancer cells and has shown an acceptable toxicity profile in adult clinical trials. Our current data substantiates its potential as an active anti-leukemic agent in currently difficult to cure pediatric leukemia subtypes. Furthermore, we provide evidence on useful drug combinations and target modulation data to characterize the molecular mechanisms and biological correlates of distinct proteasome inhibitors in pediatric leukemia. This information provides key primary data for further in vivo studies and to design effective early phase clinical trials in the near future. Disclosures: No relevant conflicts of interest to declare.

Background Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for malignant and non-malignant hematological disorders. Unfortunately, complications of HCT, primarily graft versus host disease (GVHD) and relapse of the underlying disease are substantial. In Alberta, all HCTs are performed with antithymocyte globulin conditioning and >95% of these use HLA matched donors. However, in spite of that approximately 35% Albertan HCT recipients die or suffer long-term from GVHD while another 20% die due to disease relapse. Unfortunately, strategies/treatments aiming to control GVHD in most cases lead to increase in the rate of disease relapse and infections. In the recent years, the natural killer (NK) cell genetic system has garnered substantial research interest as an immunogenetic system that significantly influences HCT outcomes. The complexity of NK cell function is modulated by a series of activating and inhibitory cell surface receptors known as the Killer Immunoglobulin-like Receptors (KIR). Here we set out to determine the influence of KIR matching between HCT donor and recipient pairs on allogenic HCT complications. Study design Hypothesizing that donor-recipient KIR gene/profile mismatch would affect the allo-HCT outcomes, we genotyped 92 (discovery cohort) and 111 (validation cohort) allo-HCT pairs by Luminex-based rSSO method. The KIR genotypes were classified into AA and Bx genotypes on the basis of KIR gene constitution of the individual. Donor (D) and recipients (R) were ‘matched' for KIR genotypes when both carried either AA (two copies of group A haplotypes) or B/x (at least one copy of group B haplotype) genotypes. Effect of KIR matching on significant GVHD (described as grade 2-4 acute GVHD or chronic GVHD needing systemic therapy) as well as disease relapse was analyzed using binomial regression and Kaplan-Meier statistics. Results As observed in both the discovery and validation cohorts, a significant protection against GVHD was conferred upon when both the donor and recipient were matched for the KIR genotypes (HR=2.224; p=0.01) without any effect on disease relapse (HR=1.098; p=0.934) (figure1). Conclusions In spite of D-R matching for major immunogenetic determinants like HLA, complications of allogeneic HCT are significant. Since GVHD represents a flip side to graft-vs-leukemia (GVL) reaction, strategies to decrease GVHD in most cases have resulted in increased relapse. Matching donor and recipients for KIR genotypes can offer a significant protection against GVHD without increasing the risk of disease relapse. Disclosures: No relevant conflicts of interest to declare.

Aim Uncontrolled reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disorder (PTLD) is one of the major complications of T-cell depleted HCT associated with a high risk of mortality. Given the fact that NK cells are first in line of defense against viral infections, and are among the earliest to reconstitute after HCT, they are deemed important in the immunopathogenesis of EBV complications. Their exact role in this process, however, remains elusive. The complexity of NK cell response is a function of a series of activating and inhibitory cell surface receptors known as Killer Immunoglobulin-like Receptors (KIR), which sense perturbations in HLA expression after viral transformation of the target cell. Here, we set out to determine whether and how KIR gene repertoire of HCT donors and/or recipients influences the development of PTLD after allo-HCT. Methods 92 HCT donor-recipient pairs and 50 healthy individuals were KIR genotyped by Luminex-based rSSO method. The KIR repertoires were classified into AA and Bx genotypes as well as into centromeric and telomeric linkage groups. PBMNCs from healthy volunteers were stimulated with EBV lysate to enumerate EBV-induced NK cell response (degranulation and/or IFN γ production). Results Donor Centromeric KIR linkage group (2DS2-2DL2-2DS3-2DL5B) was strongly associated with the incidence of PTLD (p = 0.01, OR = 7.1). Further, the number of EBV induced IFNg producing and/or degranulating NK cells were significantly lower in individuals with than without centromeric KIR linkage group. Conclusions NK cell responsiveness, a function of KIR gene repertoire has a profound effect on the development of PTLD. KIR genotype based identification of HCT recipients at high risk of developing PTLD will enable closer monitoring of EBV DNAemia and facilitate prompt therapy.

Aim Natural Killer (NK) cells, first in line of defense against tumors and infections are the earliest lymphocytes to recover after Hematopoietic Cell Transplantation. NK cell responses are regulated by activating and inhibitory receptors known as Killer Immunoglobulin-like Receptors (KIR). It is still not known which NK cell subsets elicit anti-cancer or anti-viral immune response and if target induced NK cell responses are influenced by KIR gene content. Here we assessed in healthy individuals whether different NK cell subsets elicit unique immune responses against different targets (leukemia cells or herpes viruses), and if this difference is a function of individual’s KIR gene content. Methods PBMNCs from 50 healthy donors were stimulated with a leukemic cell line (K562) and a herpesvirus (Epstein-Barr virus, EBV). A 5-colour flow cytometry based estimation of cytotoxicity (CD107a expression) and cytokine (IFN- γ ) production was performed for both regulatory (CD56 bright CD16 neg ) and cytolytic (CD56 dim CD16 pos ) NK cells. KIR gene content was obtained by Luminex based rSSO method. Results Unique target induced patterns of responses were observed with different NK cell subsets. K562 cells induced degranulation with or without IFN- γ production. Contrarily, EBV induced NK cells exhibited all single (IFN- γ production and degranulation alone) and bifunctional (IFN- γ production and degranulation together) responses. In contrast to cytolytic NK cells, which in general exhibited a bifunctional response against both targets, regulatory NK cells were primarily only IFN- γ producers. High number of activating KIR genes (B/x genotypes) showed significant correlation with IFN- γ production but not with degranulation. Conclusions NK cell subsets elicit unique immune responses against different targets, some of which are influenced by the KIR gene content. Assessment of post-HCT recovery of these target-specific functional NK cell subsets and their KIR content will be important for the prediction of HCT outcomes.

Influenza is one of the most important respiratory diseases of mankind, yet scant data exist concerning the frequency and clinical course of influenza in severely immunocompromised adults. From October 1993 to September 1994, we cultured the respiratory secretions of all adults with leukemia who were hospitalized with an acute respiratory illness at The University of Texas M.D. Anderson Cancer Center in Houston. During a 9-week period from 29 November 1993 to 29 January 1994, influenza virus type A (H3N2) was isolated from 15 (33%) of these 45 hospitalized adults. Twelve (80%) of the cases of influenza were associated with pneumonia, and four patients (33%) with pneumonia died. Patients who died tended to have received chemotherapy more recently and to be more myelosuppressed. Autopsy examination in two cases revealed histopathologic changes consistent with viral pneumonia. During community outbreaks, influenza is a frequent cause of serious respiratory disease in hospitalized adults with leukemia. Effective prophylactic and therapeutic regimens need to be defined for immunocompromised patients.

Abstract 3573 Introduction: Recent studies have shown that cell cycle events are tightly controlled by complex and shared activities of a select group of kinases. Among these, Polo like kinases (Plk) are regulatory mitotic proteins that are overexpressed in several types of cancer and are associated with poor prognosis. Consequently, inhibitors for these kinases are currently being evaluated as anticancer drugs in Phase I and Phase II clinical trials. However, details of the mechanisms by which the interference of selective regulators of mitosis lead to cell death is currently unavailable. We describe experimental studies to further understand this process and to investigate the activity, drug combinability and target modulation of Plk inhibition in refractory pediatric leukemia cells. Methods: Cell lines derived from relapsed pediatric leukemia patients were used (n=12). These cells carry the spectrum of moleular abnormalites found in patients including MLL gene rearrangemnet with various fusion genes (Bcr-Abl), and biphenotypic features. Leukemia cells were incubated with increasing concentrations of the Plk1 inhibitor volasertib (BI 6727, Boehringer Ingelheim) and after four days in culture, cell growth characteristics including cellular morphology, ploidy status and cell survival were quantified by automated quantitative microscopy. IC50 values and combination indices were calculated according to the method of Chou and Talalay. Activities of intracellular signaling components and regulators of distinct apoptosis and autophagy pathways were identified by Western blot analysis. Results: Volasertib inhibited the growth of all leukemia cell lines tested, as well as primary specimens from leukemia patients, at sub-micromolar concentrations (IC50 from 1 × 10−6 to 1 μM). Microscopic and ultrastructural analyses revealed a corresponding increase in the number of cells displaying abnormal phenotype and mitotic catastrophe, a characteristic of Plk inhibition. Plk inhibition also resulted in an initial upregulation of Aurora-A and Aurora-B expression and the phosphorylation of Aurora-B and Aurora-C. However, sustained Plk inhibition led to time dependent dephosphorylation of Aurora-A. In addition, Plk inhibition resulted in Caspase-9 cleavage and decreased expression of autophagy related proteins (Atg3). Overall, significant synergy was observed when AK inhibitors were combined with Plk inhibition (CI = 0.02 – 0.8). Conclusions: We provide experimental data to show that Plk1 inhibition by volasertib significantly decreases the growth and survival of refractory pediatric leukemia cells in vitro. We also show synergy between AK and Plk inhibition in leukemia cells and that an effective target modulation by Plk inhibition leads to alterations in intracellular AKs in a time dependent manner. Our data provide relevant information regarding the relationship between the two kinases and the intracellular changes that can be used as biological correlates for therapeutic activity in future clinical studies. These findings provide initial pre-clinical evidence for Plk as a potentially effective target in pediatric leukemia therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3074 Children with high-risk NB often receive using multiple treatment modalities including autoHCT. Disease remains the most common cause of treatment failure and the most recent cooperative group studies report survival rates approaching 45%. Prior reports showed comparable outcomes of patients with NB receiving auto- and allo-transplants, despite differences in relapse risk and treatment-related mortality (TRM). To update these data the CIBMTR conducted a retrospective review of 143 transplants reported 1990–2007 at 61centers, the largest group reported to date. For this analysis, patients were categorized into 2 groups for comparison: those without (Group 1; n=97) and those with a prior auto HCT (Group 2; n=46). Of the patients in Group 1, 31% were in first remission, 25% in partial response (PR) or very good PR (VGPR), and 24% with no response or with persistent/progressive disease. Of the patients Group 2, 17% were in first remission, 26% in PR or VGPR, and 28% with no response or with persistent/progressive disease. Median ages at alloHCT were similar for Group 1 (5y, range Univariate analysis by donor-type also indicated higher relapse rates after unrelated donor transplants and lower relapse rates after HLA-mismatched related transplants compared with HLA-identical related donor transplants (p Our analysis indicates that alloHCT can cure some NB patients, with lower relapse rates and improved survival in patients without a history of autoHCT compared with those patients who had undergone auto HCT first. AlloHCT for patients after autoHCT does not seem to offer benefit. However, the reasons for Group 1 patients not receiving autoHCT as part of their upfront therapy are unclear, and this group could include patients who were curable without alloHCT. Disease recurrence remains the most common cause of treatment failure while TRM is low. Future studies comparing OS after autoHCT and alloHCT for patients in CR1 should be considered. Disclosures: No relevant conflicts of interest to declare.

Abstract 4342 Preclinical Studies of Cytotoxicity, Drug Synergy and Biological Correlates of Clofarabine Against Infant Leukemia Cells. Infants with leukemia, specifically those who relapse on frontline therapy, are extremely difficult to cure and are candidates for novel therapies that induce remission, allowing them to proceed to transplantation. The unique molecular, growth and chemoresistance properties of infant acute lymphoblastic leukemia (iALL) allow for focused preclinical studies within this group. Clofarabine (Clolar), an anti-neoplastic purine nucleoside analog, has shown significant efficacy in older children with refractory lymphoblastic leukemia. Its safety profile has more recently been established in Phase I and II single-agent trials. In addition to its anti-metabolite action, clofarabine appears to disrupt the integrity of mitochondrial membranes and activates pathways of programmed cell death, adding to its theoretical potential to synergize with agents that interfere with mitochondrial integrity. Methods: Primary leukemic cells, cell lines derived from iALL patients and cell lines carrying the molecular abnormalities commonly found in iALL, were used in this study (n=10). Karyotypic abnormalities of these cells include: t(11;19) (q23;p13), t(9;11)(p21;q23) and t(4;11)(q21;q23). With respect to Flt-3 expression, cell lines demonstrating wild-type, internal tandem duplication (ITD) and over-expression phenotypes were also included in this panel. Primary infant AML samples (n=2) and the infant AML cell line TIB-202 (THP-1) containing the t(9;11)(p21;q23) rearrangement and MLL-AF9 fusion gene were also included. ALL cell lines derived from pediatric patients (n= 5) were evaluated in parallel. Stromal cells established from normal bone marrow specimens and peripheral blood mononuclear cells were evaluated under identical conditions for assessment of non-specific toxicity. An increasing concentration of clofarabine was added to leukemic and control cells (104 cells per well, in 96 well plates) cell lines. Over the following four days, cell growth inhibition was measured by the Alamar blue assay. For drug combination studies, leukemia cells were incubated with a panel of conventional and targeted therapeutic agents (n=12) alone or in combination with clofarabine (IC10 or IC25 concentrations). Growth inhibition under each condition was measured and combination indices were calculated according to established methods. Induction of apoptosis and the release of mitochondrial mediators were measured by Western blot analysis. Alteration in mitochondrial integrity was evaluated by immunocytochemistry for fluorescent labeled anti-mitochondrial Hsp70 and real-time imaging. Results and Discussion: Clofarabine inhibited growth of all iALL cells tested with IC50 values ranging from 0.1 μ M to 0.01 μ M. Primary iAML cells were found to be most sensitive to clofarabine. For iALL cell lines the highest IC50 value was found in Bel-1 cells, expressing a t(4;11)(q21;q23) karyotype. Drug combination studies showed significant synergy with 17-AAG (Hsp90 inhibitor, CI 0.7), sorafenib (CI 0.12), bortezomib (CI 0.3) and rapamycin (CI 0.2). No drug combinability was noted, with conventional alkylating agents and antimetabolites. Interestingly, the therapeutic opioid methadone (D,L-methadone hydrochloride), used extensively in the treatment of cancer pain and opioid addiction, showed significant synergy with clofarabine at low concentrations (CI 0.74, range 0.66 – 0.79 μ M). Incubation of cells with clofarabine (IC25) for 48 hours resulted in detectable activation of caspase 9 and cleavage of PARP. We demonstrate the ability of clofarabine to induce cytotoxicity against a panel of leukemia cells that carry the molecular aberrations and growth properties seen in iALL. We also present data on the biological correlates and synergistic effects of clofarabine with other anti-leukemic agents. Of particular interest is the synergy with methadone, which has been shown previously to affect mitochondrial activity in leukemia cells. Data presented in study provide key initial data to construct effective xenograft studies and to formulate a clofarabine based treatment protocol for iALL in the near future. Disclosures: Narendran: Genzyme Canada: Research Funding.

Abstract 680 Cytogenetics play an essential role in determining the prognosis of patients with AML. However, there is still no validated cytogenetics grouping scheme that specifically applies to patients undergoing allogeneic stem cell transplantation, which hampers accurate prognostication and risk stratification. We studied 821 adult patients (median age 41, range 18–74) who underwent SCT between 1999 and 2004 for AML (excluding APL) in CR1 or CR2 and who were reported to the CIBMTR from centers with >20 patients meeting study criteria. 75% of patients received a myeloablative conditioning. 496 patients had a normal karyotype. The primary cytogenetics reports were manually reviewed for 92% of the patients with an abnormal karyotype. We compared the ability of the 6 existing grouping schemes (MRC, CALGB, EORTC/GIMEMA, SWOG/ECOG, DFCI, and Monosomal Karyotype (MK) classifications) to stratify patients, using both the Akaike Information Criterion in multivariable models and the C-statistic. Among all existing schemes, the DFCI system provided a marginally superior stratification for overall and leukemia-free survival. We also built a new classification using individual cytogenetic abnormalities in a Cox model that included other significant covariates (performance status, therapy-related disease, conditioning intensity, graft source, donor match, duration of CR1, and gender match). This CIBMTR scheme (see Table), which is similar to the DFCI scheme except for the inclusion of patients with t(8;21) in the intermediate group, could stratify patients into 3 groups with similar treatment-related mortality but significantly different overall survival, leukemia-free survival, and incidence of relapse. This scheme appeared to apply to both patients in CR1 and in CR2 (see FiguresF2). This transplant-specific scheme could be adopted for prognostication purposes and to stratify patients with karyotypic abnormalities entering transplantation clinical trials. Future studies may clarify the relative outcome of patients with t(8;21) and refine this scheme with the inclusion of molecular abnormalities. Disclosures: No relevant conflicts of interest to declare.

Objective To report a case of a pyosalpinx presenting as an ectopic pregnancy. Subject A 31-year-old patient was admitted with severe pain in the right iliac fossa 14 days after a curettage of the uterine cavity for missed abortion following 10 weeks of amenorrhoea. Intervention An ultrasound scan showed a mass next to the right ovary with no evidence of retained products of conception. The β-hCG level was 1258 IU l−1. A laparoscopy suggested an ectopic pregnancy in the distal end of the right tube. A salpingectomy was performed and the histopathological findings were of acute salpingitis. Conclusion We describe a pyosalpinx after curettage of the uterine cavity, with high β.-hCG levels from an intrauterine pregnancy, which indicated the wrong diagnosis of an ectopic pregnancy.

Abstract 3078 Poster Board III-15 Leukemia in children less than 1 year of age confers a poor prognosis, despite intensification of therapy. These leukemias possess unique biologic characteristics including the presence of mixed-lineage leukemia (MLL) gene rearrangement and high expression of Fms-like tyrosine kinase 3 (FLT3). AT9283, a potent inhibitor of Aurora A and B kinases, JAK2, JAK3, and mutant Abl Kinase, has demonstrated inhibition of multiple solid tumor cell lines in vitro and in mouse xenograft models. Aurora kinase inhibition has been shown to inhibit cancer cell growth by interfering with the mitotic apparatus. We investigated the activity of AT9283 against cell lines derived from refractory infant leukemia cells to identify its efficacy in a future treatment protocol. Method Five cell lines derived from infant leukemia cells were used (ALL: BEL1, KOPN8, KCCF2, B1 and AML: TIB202). We also included the cell line SEM that was derived from a 5 year old child with t (4;11) MLL-AF4 preB-ALL. Normal bone marrow stromal cells were used to evaluate cytotoxicity against non-malignant cells. AT9283 was provided by Astex Therapeutics Ltd. (Cambridge, UK). Approximately 1×104 cells per well were seeded in 96-well plates and incubated with increasing concentrations of AT9283, alone or in combination with a panel of conventional and novel therapeutic agents. After four days, cell survival was measured by Alamar blue assay and IC50 values and combination indices were calculated. Stem-like cells were quantified by the distribution of ALDH bright cells by Aldefluor assay (Stem cell technologies) and characterized by conventional clonogenic assays. Alterations in cell-signaling pathways and survival proteins were measured by Western blot analysis using total and phospho-specific antibodies. Results AT9283 inhibited the growth of all five cell lines with a 10 fold variation in IC50 within cell lines (IC50 range, 0.1 to 0.01 μM). There was a corresponding increase in the number of cells displaying a polyploid phenotype, an effect of aurora kinase inhibition. No significant cytotoxicity against bone marrow stromal cells was seen under the experimental conditions used in this study (IC50 > 10 μM). Changes in the activation and expression of a variety of intracellular proteins were noted, including the down regulation of activated ERK1/2, MYC and AKT within 10 minutes of exposure to the agent. An increase in the activated form of RAF and ATF2 was observed immediately after drug exposure. Importantly, a significant decrease in the level of constitutive pFLT-3 was demonstrated. A concurrent increase in cleaved PARP was also noted, indicating the initiation of apoptosis. In combination studies, the HDAC inhibitor Apicidin showed synergy across all cell lines (CI range: 0.07 to 0.62). A decrease in ALDH bright stem-like cells was observed in a dose dependent manner, up to 50% over 24 hours at IC50 concentrations. Conclusions Our in vitro studies show that AT9283 significantly decreases the growth and survival of infant leukemia cell lines. Importantly, AT9283 potently induces FLT3 de-phosphorylation, inhibiting a critical growth stimulatory pathway of infant ALL cells. We have identified changes in a number of signaling and apoptotic molecules that can provide a panel of markers for biological correlative analysis for drug activity in vivo. Also, the drug combination studies demonstrate the potential of HDAC inhibition to synergize with the activity of this agent. Finally, the effect on stem-like cells provides a rationale and critical preclinical data for the formulation of an effective clinical trial for the treatment of infants with refractory ALL. Disclosures Squires: AstexTherapeutics Ltd: Employment.

Introduction: Currently, patients with refractory Hodgkin lymphoma (HL) or those who relapse multiple times have an extremely poor prognosis. Even patients who do not relapse often experience late toxicities, including secondary cancer, heart failure, pulmonary dysfunction, and infertility. Therefore, new agents and novel therapeutic approaches are urgently needed. Anti-apoptotic proteins such as Bcl-2 and Bcl-xl have been found to be associated with the growth and survival of Hodgkin Reed–Sternberg cells and thus carry the potential to be effective targets for therapeutics. In this study we report the in vitro cytotoxicity, biological correlative findings and drug combination analysis of the novel BH3 mimetic ABT-737 [Abbott laboratories (Abbott Park, IL)] against HL cells. Materials and Methods: HL cell lines (KMH2 and HDLM2) were cultured in the presence of increasing concentrations of ABT-737 or its enantiomer control. Normal bone marrow stromal cells were used as controls for non-specific cytotoxicity. Cell growth inhibition was measured by Alamar blue assay and the induction of apoptosis was demonstrated by an annexin specific staining technique. Time and drug concentration dependent changes in proteins involved in cell survival and apoptosis were investigated by Western blot analysis. The ability of ABT-737 to influence the anti-lymphoma activity of a panel of twenty distinct chemotherapeutic agents was evaluated by drug combination and cell growth inhibition studies. Combination indices (CI) were calculated to identify therapeutic enhancement of different agents in the presence of Bcl-2 inhibition by ABT-737. Results: ABT-737 showed significant dose-dependent cytotoxicity and apoptotic activity against HL cells with an approximate IC50 of 1mM with maximum cell death occurring at about 5mM. At the molecular level, increased levels of cleaved caspase3 and PARP, as well as annexin positivity, were noticeable within three hours of treatment with the agent. Drug combination studies have shown the ability of Bcl-2 inhibition to synergize with novel therapeutic agents that target histone deacetylase function (Apicidin, CI 0.35), Hsp90 stability (17-AAG, CI 0.3) and the activity of specific receptor tyrosine kinases (Sorafenib, CI 0.7 and Sunitinib CI 0.3 for HDLM2 and CI 0.9 for KMH2). Among conventional anti-neoplastic agents, ABT-737 showed significant synergistic activity with irinotecan and oxaliplatin (CI 0.6). Importantly, treatment with ABT-737 decreased the expression of the critical HL cell growth promoter, NF- B as determined by band densities on Western blot analysis: a two fold decrease in KMH2 and a five fold decrease in HDLM2 cells. Specific target modulation was demonstrated by changes in key apoptosis and cell survival regulators such as Bcl-XS/L, Bcl-X, p53 and survivin by two to 10 fold decreases in Western blots. Discussion: Data presented in this study support the hypothesis that Bcl-2 family of proteins can be an effective target for therapeutics in HL. We have shown that the BH3 mimetic ABT-737 induces apoptosis in these cells, characterized by the modulation of key components of cell growth and survival pathways. In addition, we have identified distinct classes of anti-lymphoma and anti-neoplastic agents whose activities are enhanced by concurrent inhibition of Bcl-2. These findings provide the rationale for further evaluation of ABT-737 and the subsequent clinical development of a targeted anti-Bcl-2 therapy for refractory Hodgkin lymphoma.

Patient specific adverse reactions are common when HPC, Cord Blood (HPC-C) units, cryopreserved in dimethylsulfoxide (DMSO) are warmed to 37°C and infused into pediatric patients. Adverse reactions, including hemodynamic instability, fever, chills, nausea, cardiomyopathy, unconsciousness, and even death have been described in the pediatric population. Additionally, hematopoietic stem cells (HSC) cryopreserved in DMSO have limited viability upon thawing due to the cellular cytotoxicity of DMSO, resulting in the potential for significant loss of cells available for transplantation and subsequent engraftment. Although DMSO is essential as a cryoprotectant for long-term preservation of hematopoeitic stem cells, standardized techniques are required to mitigate the cellular cytotoxicity and acute onset adverse events witnessed in unwashed/undiluted cord blood transplantation. Immediate mixing of thawed cord blood units with a hypertonic reconstitution solution can ameliorate many of these problems by restoring the osmolarity of the suspension, promoting colloidal-osmotic intracellular equilibrium, and reducing DMSO concentration in the infused product. In general, studies examining the benefits of dilution and/or washing of HPC-C prior to infusion have largely been confined to adult populations with varying results. Since pediatric patients often experience more significant adverse reactions, even when DMSO concentrations in the infused unit are well below the recommended maximum of 5 mL of 20% DMSO/kg body weight, examination of effective and robust techniques for minimizing these effects in the pediatric patient is crucial. The studies presented demonstrate successful utilization of diluted HPC-C products in HSC transplants in pediatric patients. HPC-C products were either infused immediately after thaw or infused after dilution with a solution of 8% Gentran-40 and 5% HSA (ReCon solution). Dilution of HPC-C products immediately after thaw appears to ensure maximal HSC recovery and viability--essential for transplant and engraftment. In experimental studies, improved HPC recovery and viability was observed in all diluted HPC-C products as compared to undiluted products. As well, the number and viability of CD34+ cells remains constant for >24 hours post thaw when HPC-C products are diluted in ReCon solution. In contrast, thawed and undiluted HPC-C units experience a significant reduction in number and viability of CD34+ cells 15–30 minutes post-thaw. Most importantly, pediatric patients displayed reduced adverse reactions when infused with diluted HPC-C products as compared to undiluted products. Dilution of HPC-C products with ReCon solution did not affect engraftment of neutrophils and/or platelets in these patients. In a specific case study, a diluted HPC-C product resulted in engraftment when a double-cord transplant with undiluted HPC-C products did not. As well, the negative adverse reaction experienced by the patient following infusion of undiluted product was not experienced when the diluted HPC-C product was infused. In the pediatric HSC transplant population it is essential not only to ensure optimal recovery and potency of HSCs in the transplant graft but also to alleviate adverse reactions. Application of HPC-C dilution protocols employing a solution of Gentran-40 and HSA provide an effective, safe, and efficient strategy for diminishing DMSO related toxicities in the pediatric HSCT patient.

We present two cases of young patients with vague abdominal pain. During laparoscopy, multiple violin string-like adhesions in the upper abdomen were seen. This appearance is thought to be caused by Chlamydia infection and this perihepatitis is called the Fitz-Hugh–Curtis syndrome. Laparoscopic intervention using endoscissors to cut the adhesions was performed. Both patients made an uneventful recovery. A 14-day course of doxycycline for the patients and their partner is the optimal medical treatment.

The optimal treatment for children with acute lymphoblastic leukemia (ALL) in second remission after an isolated central nervous system relapse is unknown. To address this question, we compared outcomes in 149 patients enrolled on Pediatric Oncology Group chemotherapy trials 9061 (n=79) and 9412 (n=70) and HLA-matched sibling transplant recipients (n=60) reported to the Center for International Blood and Marrow Transplant Research. Patients received treatment between 1990 and 2000. Median follow-up was 8 years and 9 years after chemotherapy and transplantation, respectively. Groups were similar with respect to sex and leukocyte count at diagnosis. Chemotherapy recipients were younger at diagnosis (5 vs. 7 years) and more likely to have had a longer duration of first remission (duration of first remission ≥18 months: 70% vs. 48%). All transplant recipients received bone marrow grafts and 83% received a total body irradiation containing preparatory regimen. The median time to transplantation after achieving a second remission was 2.5 months (range

INTRODUCTION: The use of peripheral blood as a source of stem cells for pediatric allogeneic stem cell transplantation remains controversial. We present a retrospective analysis evaluating the safety and efficacy of allogeneic peripheral blood stem cell transplantation (PBSCT) compared to allogeneic bone marrow transplantation (BMT) for the treatment of acute leukemia at our centre. The impact of ATG added to the treatment regimen was also evaluated in this analysis. METHODS: A chart review was conducted on all pediatric patients who underwent an allogeneic stem cell transplant for acute leukemia between April 1991 and August 2003 at the Alberta Children’s Hospital. All patients who received stem cells from HLA matched or one-antigen mismatched donors were included in this analysis. Data were analyzed using SPSS. RESULTS: 85 children (ages 9 months to 17 years) met study criteria. 24 patients underwent a PBSCT while 61 patients, a BMT for the treatment of either ALL (n=54) or AML/MDS (n=31). Median follow up time was 5.9 years. Six of 24 patients in the PBSCT group, and 30 of the 61 patients in the BMT group received ATG (p=.042). There was a trend towards increased overall survival (OS) at 5 years following PBSCT (p=.078). Disease free survival (DFS) at 5 years following PBSCT and BMT was similar (p=.297). Subgroup analysis found no difference in OS or DFS between PBSCT and BMT in patients receiving (p=.221, p=.820) or not receiving ATG as part of the conditioning regimen (p=.448, p=.820). Chronic GVHD was increased following PBSCT (45.8%) compared to BMT (19.7%) (p=.015). The rate of severe cGVHD, however, was not significantly different (25% vs. 11.5% for PBSCT and BMT, p=.119) between the two groups. No patients receiving ATG prior to PBSCT developed cGVHD (p=.009). Addition of ATG to the transplant regimen had no impact on the rate of cGVHD following BMT (p=.221). There was no increase in mortality due to cGVHD following PBSCT (p=.243) or BMT (p=.865). Although, there was a trend to improved DFS in patients who developed cGVHD following PBSCT, this was not statistically significant (p=.057). Despite a statistically significant increase in TRM found in all patients with cGVHD at one year post-transplantation (p=.048), there was no difference in TRM between the PBSCT (4.2%) and BMT (14.8%) groups (p=.173). CONCLUSION: Allogeneic PBSCT is a safe and effective alternative to allogeneic BMT for the treatment of acute leukemia in the pediatric population. Although there is an increased rate of cGVHD following PBSCT this does not appear to influence OS, DFS or TRM in our analysis. Addition of ATG into the transplant regimen decreases the occurrence of cGVHD after PBSCT. Studies to further assess the role of ATG in PBSCT are warranted.

Background:APC is an alternative to conventional diathermy and ND:YAG laser for hemostasis of large surface bleeding. Features of benefit for small children include limitation of maximum penetration to a tissue depth of 3mm and no tissue vaporization, reducing the risk of perforation, and ease of application through small flexible endoscopes (2.2 mm channel). Case report: A nine month old male infant presented with melena, anemia and thrombocytopenia. Melena was not decreased by platelet infusions. Upper endoscopy revealed diffuse angioma in the stomach with bleeding but none in the duodenum or esophagus. Scattered bleeding lesions were seen in the colon. Epinephrine (1:1000) injections in the stomach, controlled bleeding briefly (24hrs) on 4 occasions.The 5th injection was complicated by tachyarrhythmia, hypertension and increased bleeding.e-aminocaproic acid, steroids and somatostatin were ineffective. Bleeding worsened over 2 months up to an RBC requirement of 30 cc/kg/day.The number of gastric lesions increased, especially in the fundus.APC was performed under general anesthesia with a Pentax EG2901 endoscope. A flexible APC probe was passed through the instrument channel to within 3-5 mm from the lesion. Argon flow at 0.7-0.9 l/min and coagulation current adjusted between 40-50 watts power with small bursts was sufficient for hemostasis and ablation of nonbleeding angiectasias. Melena stopped for 10-12 days following the procedure which was repeated on 5 occasions at 14 day intervals. New lesions were noted at successive endoscopies until treatment with interferona-2b reduced the appearance of new lesions.Complications included passage of a mild electric current once while the baby was being touched during the procedure and scar formation at the site of a fundic ulcer resulting from repeated ablation.Conclusion:Argon plasma coagulation is safe and effective in the management of multiple mucosal bleeding lesions in the GI tract in infants.

In 579 singleton breech deliveries the total perinatal mortality rate was 10.4% and the corrected perinatal mortality rate was 0.67%. There were 452 babies delivered vaginally (78.1%) and 127 delivered by cesarean section (21.9%). The perinatal mortality rate was very high in the premature breech delivery and in the low-birth-weight infant. Cesarean section should be used to avoid a traumatic vaginal delivery and it should be used more liberally in the mature breech, but it is unlikely that cesarean section rates in excess of 50% will significantly reduce the perinatal mortality mortality rate. More information is required as to whether extending the indications for cesarean section in the low-birth-weight infant will further reduce the perinatal mortality rate.