Acute Lymphoblastic Research Articles

L-asparaginase isolated from Streptomyces ansochromogenes promotes Th1 profile and activates CD8+ T cells in human PBMC: an invitro investigation.

A new L-asparaginase produced by Streptomyces ansochromogenes UFPEDA 3420 actinobacteria was used in this study against human lymphocyte cultures to evaluate the immunological profile induced by this enzyme. Cultures of lymphocytes were stimulated with S. ansochromogenes L-asparaginase, and cytotoxicity, cell viability, cell stimulation and cytokine production were analysed. This new S. ansochromogenes L-asparaginase induced activation and proliferation of the TCD8+ lymphocyte subset and produced higher TNF-α, IFN-γ, IL-2 and IL-10 levels in a 24-h assay. Streptomyces ansochromogenes L-asparaginase is a promising molecule to be used in invivo models and to deepen preclinical tests against acute lymphoblast leukaemia. L-asparaginase is an indispensable component of the chemotherapeutic treatment of acute lymphoblast leukaemia (ALL) and acute myeloid leukaemia (AML). Currently, drugs such as Asparaginase® , Kidrolase® , and Elspar® and Erwinase® are efficient against leukemic disease, but promote immunosuppression and other side effects in human organisms. Our purified S. ansochromogenes L-asparaginase showed promissory results inducing, invitro, higher immunostimulation in human PBMC, especially in T CD8+ lymphocyte subsets.

Modulation of cytokine production from cultured mononuclear cells of leukemia patients by Hypericum triquetrifolium Turra methanolic extract

The effect of Hypericum triquetrifolium Turra (Family: Hypericaceae) methanolic extract on an in vitro cytokine release (IL-2, IL-4, IL-10, IL-17A and IFN-γ) from cultured mononuclear cells was determined. The cells were obtained from acute lymphoblastic (ALL) and acute myelogenous leukemic (AML) Iraqi patients. Two concentrations (1.146 and 1.719 µg/ml) of the extract were tested. They were correspondent to 20 and 30%, respectively of the plant IC50 (5.73 µg/ml). Chemical analysis of the extract showed its richness in flavonoids (115.73 µg/ml). Assessments of cytokine levels in supernatants of cultured cells revealed that ALL, AML or control cells responded differently to the plant extract in their production of IL-2, IL-10, IL-17A and IFN-γ, but leukemic cells were better than control cells in their response, while there was no effect on IL-4 production. The results suggested that H. triquetrifolium methanolic extract exerted immunomodulatory effects on cultured cells.

Molecular Study of Parvovirus B19 Infection in Children with Acute Myeloid Leukemia

Background:Parvovirus B19 is a common viral infection in children. Nearby evidences are present about its association with acute leukemia, especially acute lymphoblast leukemia. Nevertheless, scanty reports have discussed any role in acute myeloid leukemia (AML).Purpose:To evaluate the frequency of virological markers of B19 infection including its DNA along with specific immunoglobulins G (IgG) and M (IgM) among children with newly diagnosed AML. Besides, describing the clinical importance of Parvovirus B19 infection in those patients.Patients and methods:A case-control retrospective study was conducted on 48 children recently diagnosed with AML before and during chemotherapy induction and 60 healthy control. Specific serum IgM and IgG levels were determined by enzyme linked immunosorbant assay (ELISA) and DNA detection by polymerase chain reaction (PCR).Results:Parvovirus DNA was detected in 20 patients with AML. IgM was found in sera of four patients and one case had positive DNA and IgG (5%). Patients with recent parvovirus B19 infection had a significantly reduced hemoglobin levels, RBCs counts, platelet counts, neutrophil counts and absolute lymphocytosis (p=0.01, p=0.0001, p=0.01, p=0.02, p=0.0003, respectively). There were no clinical findings with statistically significant association to recent infection. Half of the patients with AML had positive PCR and/or IgM for parvovirus B19. Among children with AML under chemotherapy, there were reduced hemoglobin levels (P=0.03), reduced platelet counts (P=0.0001) and absolute neutropenia (mean±SD, 1.200 ±1.00) in those with parvovirus B19 infection. More than half of patients with parvovirus B19 (72.2%) had positive PCR and/or IgM and 36.4% of them had positive IgG.Conclusion:This study highlights that parvovirus B19 is common in children with AML either at diagnosis or under chemotherapy. There are no clinical manifestations that can be used as markers for its presence, but hematological laboratory findings can provide evidence for infection in the presence of anemia and neutropenia. Detection of parvovirus B19 by combined molecular and serological markers is required in such patients for accurate diagnosis.

Low-dose decitabine enhances chidamide-induced apoptosis in adult acute lymphoblast leukemia, especially for p16-deleted patients through DNA damage.

To investigate the combined action of decitabine (DAC) with chidamide (CS055) on acute lymphoblastic leukemia (ALL) cells. ALL cell lines as well as primary cells from 17 ALL patients were subjected to different treatments and thereafter cell counting Kit-8 (CCK-8) assay, flow cytometry and western blot were employed to determine IC50, apoptosis and checkpoint kinase 1 and γH2A.X expression. Low-dose DAC combined with CS055 could effectively kill ALL cells by the reduction of cell viability and induction of apoptosis. This was also observed in primary cells from 17 ALL patients, especially for those with p16 gene deletion. Suppression of checkpoint kinase 1 phosphorylation and upregulation of γH2A.X expression was demonstrated to participate in DAC plus CS055-induced apoptosis. Low-dose DAC could enhance chidamide-induced apoptosis in adult ALL, especially for patients with p16 gene deletion through DNA damage.

Abstract LB-084: Histone chaperone HIRA is required for Setd1a H3K4 methylation recruited by TAL1 oncoprotein in leukemogenesis

Abstract TAL1/SCL is a hematopoietic specific member of the basic Helix-Loop-Helix (bHLH) family of transcription factors important for the development of all hematopoietic lineages and pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), which plays a coordinating role in establishing the proper chromatin dynamics and gene regulation. We have previously demonstrated that TAL1 associated with H3K4 methyltransferase SETD1A, which mediated epigenetic regulation that regulates hematopoietic transcription and differentiation programs during hematopoiesis and leukemogenesis. We now find that a novel interaction between TAL1 and histone cell cycle regulation defective homolog A (HIRA) histone chaperone. TAL1 directly interacts with HIRA, which is not only essential for promoter H3K4 hypermethylation by Setd1a, but is also critical for the transcriptional activation of target genes mediated by TAL1 and Setd1a in T cell acute lymphoblast leukemia (T-ALL) Jurkat cells, such as CDK6 and NKX3.1. Our data revealed a collaborative role by which transcription factor-directed recruitment of histone chaperone and histone modification enzyme can lead to chromatin structure alterations at specific promoters in leukemogenesis. Citation Format: Ying Li. Histone chaperone HIRA is required for Setd1a H3K4 methylation recruited by TAL1 oncoprotein in leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-084. doi:10.1158/1538-7445.AM2017-LB-084

Classification of acute lymphoblastic leukaemia using hybrid hierarchical classifiers

In current consequence of haematology, blood cancer i.e. acute lymphoblastic leukemia is very frequently founded in medical practice, which is characterized by over activation and functional abnormality of bone marrow. The abnormality is identified through physical examination with a screening of blood smears. However, this method is error prone and labor intensive task for haematologist. Hence, haematologist needs a specific computer aided diagnostic system (CAD) that can deal with these limitations of prior systems and capable of discriminating immature leukemic cells from mature healthy cells. Thus, this work addresses the problem of segmenting a microscopic blood image into different regions, and then further analyzes those regions for localization of the immature lymphoblast cell. Further, it investigates the use of different geometrical, chromatic and statistical textures features for nucleus as well as cytoplasm and pattern recognition techniques for sub typing immature acute lymphoblasts as per FAB (French--- American --- British) classification. This can facilitate haematologist for acquiring essential information about prognosis and for an appropriate cure for leukemia. The exhaustive experiments have been conducted on 260 microscopic blood images (i.e. 130 normal and 130 cancerous cells) taken from ALL-IDB database. The proposed techniques consisting of the segmentation module used for segmenting the nucleus and cytoplasm of each leukocyte cell, feature extraction module, feature dimensionality reduction module that uses principal component analysis (PCA) to mapped the higher feature space to lower feature space and classification module that employs the standard classifiers, like support vector machines, smooth support vector machines, k-nearest neighbour, probabilistic neural network and adaptive neuro fuzzy inference system.

LEUKEMIA LIMFOBLASTIK AKUT PADA DEWASA DENGAN FENOTIP BILINEAGE (LIMFOID-B DAN T)

In this report we describe a patient with adult acute lymphoblastic leukemia with bilineage phenotypic. He was found to have was found to have massive right pleural effusion with mediastinal shift to the contra lateral side. There was also a smaller left pleural effusion. He had multiple bilateral cervical lymphadenopathy, tense ascites and bilateral pedal oedema up to the shins. He was otherwise clinically stable. His full blood count on admission showed Hb of 11.4 g/dl, platelets of 59 X 109/L and WBC of 12.99 × 109/L with blasts of 26%. His renal function was normal with a creatinine of 107 micromoles/L. Bone marrow trephine biopsy showed features consistent with acute lymphoblast leukemia-L1. Flow cytrometry of his blood was suggestive of bilineage phenotypic acute lymphoblast leukaemia. It showed a single population of blasts (about 31%) which expressed cCD3+, CD4-, CD7+, CD5-, CD19+, CD34+, TdT+, cytoplasm IgM, CD79a+ and 30% are CD10+, and there was aberrant CD33+ expression with no evidence of MPO or CD117 expression. Cytogenesis of the bone marrow trephine biopsy showed numerical and structural abnormalities in nine out of the seventeen cells analysed. These abnormalitiesare 43~47,XY, add (1)(p34.2), add(2)(p13), i(17)(q10), +21[cp9].

Role of HLA match on results of hematopoietic stem cell transplantations from unrelated donors in children with acute leukemia and bone marrow failure syndromes

BackgroundIn case of the lack of matched family donors (MFD), hematopoietic stem cell transplantation (HSCT) from unrelated donor (UD) is an established procedure for many acquired and congenital disorders of the hematopoietic system, including malignancies and bone marrow failure (BMF) syndromes. ObjectiveThe analysis of the results of HSCT in patients with acute leukemia or BMF syndromes from UDs with respect to human leukocyte antigen (HLA) match. Patients and methodsA total number of 97 of HSCT from UDs performed in single center between 2007 and 2015 in children and adolescents with acute lymphoblastic (ALL) or myeloblastic leukemia (AML) and BMF syndromes were included into this analysis. HLA match between donor and recipient was analyzed at the allele level and classified as 10/10, 9/10 or 8/10. Data were compared to results of 56 MFD-HSCTs. Probability of overall survival (pOS) was given for 3-year and 1-year (as required by JACIE standards) time periods. ResultsThe mean survival for all patients estimated by Kaplan–Meier method was 4.8 years (95%CI=4.1–5.5 years). The 3-year pOS after all UD-HSCT was 0,60±0,05, and with respect to 10/10, 9/10 and 8/10 HLA match: 0,61±0,06; 0,59±0,09 and 0,60±0,22, respectively (ns). In patients with AML, 3-year pOS reached 52%, 60% and 60%, respectively. In patients with ALL, 3-year pOS was 73% and 62% (ns) for 10/10 and 9/10 HLA match, respectively, while for BMF syndromes 86% and 57% (ns), respectively. ConclusionCurrent data suggest that results of mismatched and matched UD-HSCT in children with acute leukemia might be comparable.

Molecular pathway activation features of pediatric acute myeloid leukemia (AML) and acute lymphoblast leukemia (ALL) cells.

Acute lymphoblast leukemia (ALL) is characterized by overproduction of immature white blood cells in the bone marrow. ALL is most common in the childhood and has high (>80%) cure rate. In contrast, acute myeloid leukemia (AML) has far greater mortality rate than the ALL and is most commonly affecting older adults. However, AML is a leading cause of childhood cancer mortality. In this study, we compare gene expression and molecular pathway activation patterns in three normal blood, seven pediatric ALL and seven pediatric AML bone marrow samples. We identified 172/94 and 148/31 characteristic gene expression/pathway activation signatures, clearly distinguishing pediatric ALL and AML cells, respectively, from the normal blood. The pediatric AML and ALL cells differed by 139/34 gene expression/pathway activation biomarkers. For the adult 30 AML and 17 normal blood samples, we found 132/33 gene expression/pathway AML-specific features, of which only 7/2 were common for the adult and pediatric AML and, therefore, age-independent. At the pathway level, we found more differences than similarities between the adult and pediatric forms. These findings suggest that the adult and pediatric AMLs may require different treatment strategies.

Survival trends in childhood chronic myeloid leukaemia in Southern-Eastern Europe and the United States of America

AimTo assess trends in survival and geographic disparities among children (0–14 years) with chronic myeloid leukaemia (CML) before and after the introduction of molecular therapy, namely tyrosine kinase inhibitors (TKIs) in Southern-Eastern European (SEE) countries and the USA. MethodsWe calculated survival among children with CML, acute lymphoblastic (ALL) and acute myeloid leukaemia (AML) in 14 SEE (1990–2014) cancer registries and the U.S. Surveillance, Epidemiology and End Results Program (SEER, 1990–2012). We used Kaplan–Meier curves and multivariate Cox regression models to calculate hazard ratios (HRs) with 95% confidence intervals (CIs). ResultsAmong 369 CML cases, substantial improvements were noted in 2-year survival during the post-TKI (range: 81–89%) compared to pre-TKI period (49–66%; HR: 0.37, 95% CI: 0.23–0.60). Risk of death was three times higher for <5-year-old children versus those aged 10–14 years (HR: 3.03, 95% CI: 1.85–4.94) and 56% higher for those living in SEE versus SEER (HR: 1.56, 95% CI: 1.01–2.42). Regardless of geographic area and period of TKI administration, however, age seems to be a significant determinant of CML prognosis (pre-TKI period, HR0–4y: 2.71, 95% CI: 1.53–4.79; post-TKI period, HR0–4y: 3.38, 95% CI: 1.29–8.85). Noticeably, post-TKI survival in CML overall approximates that for ALL, whereas therapeutic advancements for AML remain modest. ConclusionRegistry data show that introduction of molecular therapies coincides with revolutionised therapeutic outcomes in childhood CML entailing dramatically improved survival which is now similar to that in ALL. Given that age disparities in survival remain substantial, offering optimal therapy to entire populations is an urgent priority.

The ability of multipotent mesenchymal stromal cells from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells.

The development of leukemia impairs normal hematopoiesis and marrow stromal microenvironment. The aim of the investigation was to study the ability of multipotent mesenchymal stromal cells (MSCs) derived from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells. MSCs were obtained from the bone marrow of 14 patients with acute lymphoblastic (ALL), 25 with myeloid (AML), and 15 with chronic myeloid (CML) leukemia. As a control, MSCs from 22 healthy donors were used. The incidence of cobblestone area forming cells (CAFC 7-8 d) in the bone marrow of healthy donor cultivated on the supportive layer of patients MSCs was measured. The ability of MSCs from AML and ALL patients at the moment of diagnosis to maintain normal CAFC was significantly decreased when compared to donors. After chemotherapy, the restoration of ALL patients' MSCs functions was slower than that of AML. CML MSCs maintained CAFC better than donors' at the moment of diagnosis and this ability increased with treatment. The ability of patients' MSCs to maintain normal hematopoietic progenitor cells was shown to change in comparison with MSCs from healthy donors and depended on nosology. During treatment, the functional capacity of patients' MSCs had been partially restored.

Лейкемическая инфильтрация зрительного нерва как первое проявление нейролейкемии

Extramedullary leukemic foci in acute leukemia attract special attention. They reflect the diversity of clinical manifestations of the disease, affect the severity of the patient’s condition and may influence the choice of treatment strategy. Purpose: to present a rare clinical case of leukemic optical neuropathy in a patient with acute lymphoblast leukemia. Results. Decreased visual acuity of both eyes related to the involvement of the optic nerve was one of the main complaints of the female patient during the onset of the diseases. A loose greyish tumor with blurred contours was identified on the optic nerve disc, with a large number of hemorrhages in various layers of the peripapillary retina. During ophthalmoscopy, the central vessels in the optic disc funnel could not be distinctly differentiated. Brain and orbit MRI showed intraocular masses in the area of the projection of the optic nerve disc and a lesion in the left frontal lobe. There were no signs of high intracranial pressure. After the first chemotherapy course for acute leukemia, visual acuity of both eyes increased, along with a substantial regression of tumor infiltration of the optic disc, which made it possible to visualize the border of the optic disc and the central retinal vessels. Follow-up images showed a full regression of the mass in both eyeballs. The left frontal lesion, whose origin was unclear, remained unchanged. Conclusion. An isolated lesion of the optic nerve could be a potential first sign of neuroleukemia, so it should be on the list for differential diagnosis of optic nerve disorders. First of all, leukemic optic neuropathy should be differentiated from optic nerve edema. Early diagnosis of neuroleukemia and adequate anti-tumor treatment allow for significant improvement of visual acuity and hence, for better quality of life of patients with leukemia // Russian Ophthalmological Journal, 2016; 4: 64-8. doi: 10.21516/2072-0076-2016-9-4-64-68. .

The association between RFC1 G80A polymorphism and cancer susceptibility: Evidence from 33 studies

Aberrant folate metabolism is closely related to tumorigenesis. Genetic variations in the Reduced folate carrier 1 (RFC1) may alter the progress of folate metabolism, and thereby cause the initiation and progress of the cancer. Considerable studies have performed to investigate the association between RFC1 G80A (rs1051266) polymorphism and cancer susceptibility, but the conclusions were conflicting. Therefore, we conducted a meta-analysis to reevaluate the association of RFC1 G80A polymorphism with cancer risk. PubMed and EMBASE were searched for eligible studies. The association of RFC1 G80A polymorphism and cancer risk was evaluated by the pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs). The significant association was found between RFC1 G80A polymorphism and hematological malignance susceptibility (A vs. G: OR=1.11, 95%CI=1.003-1.23, P=0.045; GA vs. GG: OR=1.18, 95%CI=1.06-1.31, P=0.002; AA+GA vs. GG: OR=1.18, 95%CI=1.07-1.29, P=0.001). Stratified analysis by ethnicity indicated that the association became more prominent among Caucasians (GA vs. GG: OR=1.28, 95%CI=1.12-1.45, P<0.001; AA+GA vs. GG: OR=1.21, 95%CI=1.08-1.36, P=0.001). In term of the cancer type, this polymorphism significantly increased the risk of acute lymphoblast leukemia (GA vs. GG: OR=1.13, 95%CI=1.001-1.28, P=0.048; AA+GA vs. GG: OR=1.28, 95%CI=1.13-1.46, P<0.001) and acute myeloid leukemia (GA vs. GG: OR=2.57, 95%CI=1.37-4.85, P=0.003). No significant association between RFC1 G80A polymorphism and overall solid cancer risk was observed, but a protective association with digestive cancer risk was found (GA vs. GG: OR=0.89, 95%CI= 0.81-0.99, P=0.030). The comprehensive meta-analysis encouraged the notion that RFC1 G80A polymorphism may play an important role in hematopoietic system malignance. These findings need further validation in the large multicenter investigations.

Alterations in the Physiology of Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Patients with Leukemia

IntroductionIn acute and chronic leukemia, the changes in the basic properties of multipotent mesenchymal stromal cells (MSCs), including morphology, immunomodulatory abilities, and the expression of various genes, were described. The aim of the investigation was to study the ability of MSCs derived from the bone marrow (BM) of patients with acute lymphoblastic (ALL), myeloid (AML) leukemia and chronic myeloid leukemia (CML) to maintain normal hematopoietic progenitor cells.MethodsThe study included 14 patients with ALL, 25 with AML and 15 with CML. All work was conducted in accordance with the Declaration of Helsinki (1964). BM was aspirated from the patient during diagnostic punctures before the treatment and for patients with acute leukemia 37 and 100 days; for patients with CML on average at the 125th day and 220th day after the start of the treatment. The BM samples of 22 healthy donors were used as controls.MSCs were derived from 5-10 ml of BM and cultivated at the density 3 х 106 cells in T25 culture flasks in aMEM with 10% FCS.The ability of MSCs from patients to maintain normal hematopoietic precursor cells - coble stone area forming cells (CAFC) was performed by the limiting dilution method. One day prior to assay, MSCs from donors and patients were explanted with 1000 cells per well of 96-well plates. As a control, the cell line MS5 supporting growth of hematopoietic precursor cells was used. On the following day, with complete medium change, BM cells from healthy donors were implanted in four serial dilutions. The frequency of CAFC in the normal BM was performed using Poisson's equation and presented as the percent of control.The total RNA was extracted from MSCs at passage 1 using the standard method. The relative gene expression levels (REL) were determined by normalizing the expression of each target gene to the levels of beta-actin and glyceraldehyde 3-phosphate dehydrogenase and calculated using the ΔΔCt method.ResultsTotal cell production of MSCs from ALL (5.6±1.5) x 106 and AML (5.4±1.0) x 106 patients decreased at the moment of diagnosis, whereas the production of MSCs from CML (7.9±1.9) x 106 patients did not differ from the donors (7.1±1.04) x 106. Reduced cell production is likely associated with a significant decrease in the expression level of FGF2, VEGF, BGLAP and SOX9 genes (Table).The ability of MSCs derived from the BM of AML patients at the onset of the disease to maintain normal CAFC (59.3±6.8) was significantly decreased (p = 0.02) when compared to donors (74.9±9.5). At the end of the first course of chemotherapy, the ability to maintain CAFC in patients' MSCs reached that of the donors(80.7±5.8); 2 months later, the CAFC frequency on MSCs from AML patients doubled (107.9±18.4) in comparison with the start of the disease (p = 0.04).The ability of MSCs derived from ALL patients to support CAFC was lower than that of MSCs of AML patients both before treatment (57.8±12) and 37 days after the start (57.2±7.4). Three months after the initiation of treatment of these patients, the ability of MSCs to maintain CAFC recovered (73.7±13.3) and reached that of the donors' MSCs.MSCs of CML patients (100.8±9.9) at the disease onset maintained CAFC better than donors' MSCs. This ability increased with treatment at 125th day (109.6±14.4) and at 220th day (169.6) after the start of the treatment. The expression levels of LIF, IGF1, IL6, CSF1 increased significantly in CML-derived as well as AML-derived MSCs, but changes were more pronounced in the case of CML.TableREL of different genes in MSCs derived from patients at the onset of the disease.GeneAMLALLCMLDonorsFGF22.8±0.2*2.0 ±0.4*4.2±0.56.0±0.9VEGF1.5±0.1*1.5±0.2*1.7±0.22.5±0.9LIF9.1±1.3*7.1±2.3*21.8±7.52.2±0.4IL612.7±1.8*11.3±4.422.5±4.8*6.2±1.8CSF13.6±0.9*2.1±1.20.96±0.1*0.7±0.1IGF11.3±0.3*1.49±0.72.6±0.5*0.6±0.1BGLAP0.6±0.2*0.8±0.4*1.7±0.42.7±0.1SOX91.2±0.1*0.9±0.3*1.2±0.21.7±0.2(*significantly different from donors)ConclusionsFunctional changes in MSCs, which are the part of BM stromal microenvironment and, in particular, a niche for the HSCs was revealed. The ability of MSCs to support CAFC is dramatically changed in patients with hematological malignancies; the nature of the functional alterations of MSCs depends on the diagnosis. In cases of acute leukemia, MSCs' ability to maintain CAFC normalized with the treatment, whereas the strengthening of this capacity was revealed in cases of CML. DisclosuresTurkina:Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis Pharma: Consultancy.

Excess Coincidences of Hematopoietic Neoplasms

Introduction:In recent years diagnostic advances, novel therapies and improved supportive care have led to growing life expectancy of patients suffering from hematologic neoplasms. Hence, the coincidence of different neoplasms may be observed more frequently challenging both clinicians as well as patients to select the most appropriate treatment approach. However, studies on this topic are rare and the issues of common etiologic factors, epidemiology, or pathogenic mechanisms are unclear yet.Aim:Compare expected and observed frequencies of coincidences of hematologic neoplasms and determine the types of coincidences.Material and Methods:Patients with co-occurring neoplasms were identified by cytomorphology, immunophenotyping, cytogenetics, and molecular genetics based on peripheral blood and bone marrow samples sent to our laboratory for diagnostic purposes (11/2011-03/2015, 3.3 yrs). Therapy-related coincidences were excluded. The ratio of the number of observed to expected coincidences was derived from published epidemiologic data (WHO, ELN, SEER). We report on coincidences of different entities distinguished by WHO criteria: acute lymphoblastic (ALL), myeloid (AML), and undifferentiated (AUL) leukemia, chronic myeloid leukemia (CML), monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia (MBL/CLL), myelodysplastic syndrome (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), monoclonal gammopathy of undetermined significance/multiple myeloma (MGUS/MM), myeloproliferative neoplasms (MPN), and mature B- and T-cell neoplasms (B-NHL, T-NHL). Analysis of B-NHL sub-categories was performed for hairy cell leukemia (HCL) as well as for follicular (FL), lymphoplasmacytic (LPL), mantle cell (MCL), and marginal zone (MZL) lymphoma.Results:Coincidences of neoplasms were detected in 828 of 46,906 patients (2%, 804 with two and 24 with three neoplasms, respectively) affecting 1% of patients diagnosed with ALL (10/981), 1% in AML (58/5,464), 29% in AUL (5/17), 1% in CML (16/1,655), 6% in MBL/CLL (382/6,811), 4% in MDS (255/6,289), 2% in MDS/MPN (40/2,716), 4% in MGUS/MM (390/9,289), 0.6% MPN (63/9,748), 8% in B-NHL (393/4,712, including 50 cases of HCL, 16 FL, 99 LPL, 26 MCL, and 112 MZL), and 8% in T-NHL (36/277).Out of theoretically possible 120 coincidences of two respective neoplasms 77 (64%) in fact were observed. MBL/CLL and MGUS/MM, respectively, co-occurred with each category. The most frequent coincidences were B-NHL with MBL/CLL (n=148), MDS with MGUS/MM (n=119), and B-NHL with MGUS/MM (n=116). Relating observed and expected frequencies corrected for prevalence and incidence revealed that coincidences were more abundantly diagnosed than statistically expected. For ALL, the highest rate was observed for coincidence with MZL (0.3/0.004, observed/expected cases p.a.), for AML with LPL (0.6/0.003), for AUL with LPL (0.3/1e-05), for CML with AUL (0.3/0.001), for MBL/CLL with LPL (11.2/0.004), for MDS with LPL (2.1/0.004), for MDS/MPN with LPL (0.9/0.002), for MGUS/MM with LPL (13/0.006), for MPN with LPL (0.6/0.006), for B-NHL with MBL/CLL (44.8/0.1), for T-NHL with HZL (1.8/0.0003), for FL with LPL (0.3/0.0005), for HZL with LPL (0.3/7e-05), for LPL with HZL (0.6/0.0003), for MCL with LPL (0.6/0.0001), and for MZL with LPL (1.2/0.0002). Table 1 lists the enrichments of all coincidences.Conclusions:The epidemiology of coincidences is notoriously difficult to study, in particular for rare entities, e.g. AUL. However, we revealed a surprisingly broad spectrum of abundant coincidences and demonstrated that their frequency is substantially higher than expected. These differences are likely due to common causal, etiologic or pathogenic mechanisms that are shared between neoplasms.Common pathways pivotal for the pathogenesis of LPL and CLL, i.e. BTK-dependent BCR signaling which can be inhibited by Ibrutinib achieving excellent response rates, were already identified. The LPL + MBL/CLL coincidence was highly enriched and additional coincidences were identified. Future studies entangling the underlying mechanisms are needed.The demonstrated excess coincidences raise the question whether more abundant preventive screenings could translate into earlier detection, more therapeutic options, and finally increased survival of affected patients. [Display omitted] DisclosuresRose:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Identification of the Genomic Rearrangements Associated with the Ex Vivo Resistance to Anthracyclines in Childhood Acute Leukemias

INTRODUCTION: Resistance to drugs is connected with several genomic changes, but the major mutations are still unknown. Daunorubicin (DNR), idarubicin (IDA), doxorubicin (DOX) and mitoxantrone (MIT) are chemotherapeutics of the anthracycline family that are commonly used to treat acute leukemias (AL). OBJECTIVE: The aim of the study was to identify changes on the genome level and compare them with ex vivo resistance to DNR, IDA, DOX and MIT among children diagnosed with acute lymphoblastic (ALL) or myeloblastic (AML) leukemia. METHODS: The in vitro drug resistance profile was determined in MTT cytotoxicity assay, which was performed on mononuclear cells taken from 44 (aCGH) and 187 (qPCR) patients. The cyanine labeled genomic DNA (Cy3 - reference, Cy5 - patient) was hybridized to comparative genomic hybridization array (SurePrint G3 Human CGH Microarray 8x60K, Agilent). Data were analyzed using bioinformatics tools, like: Feature Extraction and CytoGenomics (Agilent) and databases, such as UCSC Genome Browser and GeneCards. Additionally, we tested gene expression level by qPCR with UPL probes for 4 genes (CASP1, ITGB2, TBLX1R1, TTC28) compared to the expression level of the reference gene ACTB. The relative expression was calculated by Pfaffl method with using a REST-2009 software (Qiagen). Statistical analysis was performed using Pair Wise Fixed Reallocation Randomisation Test. RESULTS: On the basis of the MTT results, we established that leukemic blast were predominantly resistant to IDA. DNR, MIT and DOX profiles were mostly balanced. According to achieved aCGH results, we identified several frequent genomic changes detected among children with AL. Statistically significant rearrangements are presented in Table 1. In most cases, the genes, which are in the area of genetic drug resistance determinates, have regulatory roles in cell death, proliferation signal cascades, cell adhesion, migration, cell cycle and also they interact with tumor suppressors, such as p16 or p53.Table 1Most frequent genomic rearrangements among children with acute leukemia.RearrangementsDNRpDOXpIDApMITpGenesDel5q32-35.3S0.033--S0.035--FBXO38, ABLIM3, SLC6A7, ARSI, ADAM19Amp8p12-p11.21R0.039R0.023S0.035S0.047FUT10, TTI2, RNF122, DUSP26, ZNF703, ADAM9, TM2D2, HTRA4, BAG4, LSM1Del9p21.3S0.033R0.029S0.035S0.034IFNA21, MTAP, CDKN2A, CDKN2BAmp14q32.33S0.049--S0.022S0.049ADAM6, KIAA0125Del15q11.1-11.2S0.033--S0.025S0.003HERC2P3, NBEAP1, CHEK2P2Del21.11.2-p11.1S0.019--S0.025--TEKT4P2, TPTE, BAGEAmp22q11.22S0.049R0.027S0.035---S - rearrangement correlated with drug sensitivity; R - rearrangement correlated with drug resistance; p - p-valueThe results of relative expression analysis are presented in Table 2. It is known, that DOX and IDA activate NLRP3/caspase-1/IL-1β signaling cascades, which are required for effective anticancer immunity. In our experiment, we also observed the increased expression of CASP1 in DOX and IDA profiles.Table 2Relative expression level analysis.DrugsGenesCASP1ITGB2TBL1XR1TTC28DNR1,939 ↑(0,025)3,009 ↑(<0,001)0,968 (ns)0,594 (ns)IDA1,645 (ns)2,239 (ns)0,768 (ns)1,115 (ns)DOX1,934 ↑(0,008)2,365 ↑(0,002)0,758 (ns)0,907 (ns)MIT1,389 (ns)1,739 ↑(0,022)1,044 (ns)0,628 (ns)↑ - up-regulated gene; p-value in brackets; ns - not significantCONCLUSION: The analyses showed resistance to DNR, IDA, MIT and DOX are the result of many genomic changes in leukemic blasts. We assume, that rearrangements del9p21.3 and amp22q11.22 may correlate with lack of sensitivity to DOX, and amp8p12-p11.21 with resistance to DNR and DOX. The overexpression of CASP1 is strongly correlated with resistance to DNR and DOX. Expression changes of CASP1 may interfere the immune response in leukemia. Moreover, the up-regulation of ITGB2 expression is characteristic for cells resistant to DNR, DOX and MIT. Integrins participate in cell adhesion as well as cell-surface mediated signaling and plays an important role in immune response. The application of genomic rearrangements and gene expression signatures derived from resistant and sensitive cell lines may allow to develop of new strategies for cancer response prediction after anthracycline therapy. This study was supported by Grant from the National Science Centre No. DEC-2011/03/D/NZ5/05749. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3581: Rapid conversion to resistance, of a colon PDX with ret-fusion, by ponatinib treatment could potentially be attributed to the introduction of the gate keeper mutation V804M

Abstract RET encodes a receptor tyrosine kinase (RTK) involved in cellular mechanisms of proliferation, migration and differentiation. RET fusions with different genes at 10q11.2 result in constitutively activation and drive tumor development in various cancers: 10-20% of sporadic papillary thyroid cancer1, spitzoid neoplasms, CMML and 1% of lung adenocarcinomas2,3. RET fusions have been suggested to be important therapeutic targets. Ponatinib, a multi-kinase inhibitor, was approved for the treatment of Ph+ chronic myeloid leukemia (CML) and acute lymphoblast leukemia (ALL), including CML with T315I/imatinib-resistance. Ponatinib has recently been found to potently inhibit the common NSCLC fusion variant, KIF5B-RET at clinically-achievable concentrations. We have established large collection of colorectal patient derived xenografts (PDXs)4,5. Two of the poorly differentiated adenocarcinoma, CR1520 and CR2518, contain two different chromosome 10 in-frame RET-fusions, CCDC6-RET (CR2518) and NCOA4-RET (CR1520), as revealed by RNAseq and Sanger Sequencing6. Both models have also demonstrated over-expressing ret gene at mRNA levels. More importantly, they both responded Ponatinib significantly along with dephosphorylation of RET and downstream AKT, confirming these two types of Ret fusions as oncogenic drivers in these two models6. In addition, we also observed that Ponatinib treatment also rapidly drives CR2518 into its resistance (The resistant derivative is called CR2545). To investigate the underlying mechanism of the resistance, we performed RNAseq analysis of CR2545, which revealed retaining of the ret-fusion, but also the introduction of a previously described gate-keeper mutation, V804M, at ret kinase domain. V804M, a mutation frequently found in familial medullary thyroid carcinoma (FMTC), introduces bulky amino acid at position 804 and found to resistant to vandetanib. This may contribute to the resistance. However, this notion apparently contradicts to the previous observation/claim that V804M is sensitive to Ponatinib7, and ponatinib should be used to treat RET-V804M/L cancers. It is possible that the described V804M containing vandetanib resistant cells are sensitive to ponatinib, but via a non-RET mechanism for the multi-kinase inhibition nature of ponatinib; or in our case, there are other non-RET Ponatinib resistant mechanisms been introduced in CR2545 where RET is no longer the key oncogenic driver. We are currently testing other RET inhibitors and also assessing pharmacodanamic (PD) parameters to further investigate RET resistant mechanism. The further findings from the ongoing studies will be presented at the meeting. Citation Format: Mengmeng Yang, Jie Cai, Sheng Guo, Jean-Pierre Wery, Henry Qixiang Li. Rapid conversion to resistance, of a colon PDX with ret-fusion, by ponatinib treatment could potentially be attributed to the introduction of the gate keeper mutation V804M. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3581. doi:10.1158/1538-7445.AM2015-3581

Comparison Between Related T-Cell Depleted HLA-Haploidentical Stem Cell Transplantation (TCD-Haplo) and Umbilical Cord Blood Transplantation (UCBT) in Pediatric Patients with Acute Leukemia, a Eurocord, PDWP-EBMT Study

Both TCD-Haplo and UCBT are used for treating children with either acute lymphoblastic (ALL) or myeloid (AML) leukemia in need of an allograft and lacking a suitable donor. Although both these types of HSCT have been shown to be effective in curing children with acute leukemia, to date, no study has compared the outcomes of these two types of transplant.We performed a retrospective registry-based study on children (less than 18 years) with either ALL or AML, receiving, after a myeloablative conditioning regimen, a TCD-Haplo (CD34+ cell positive selection, CD3+ negative or TCR alpha/beta+ cell depletion) or single unit UCBT. Patients given pharmacological graft-versus-host disease (GVHD) prophylaxis after graft infusion in haplo HSCT were excluded.Transplants were performed from 2001 to 2012 in EBMT centers; 1067 patients received single UCBT and 266 TCD-Haplo for AML (n=478) or ALL (n=855). Median follow up was 28 (range 1-150) and 20 (range 1-152) months for UCBT and TCD-Haplo, respectively.Compared to TCD-Haplo, UCBT recipients were younger (median age 5.96 years vs 9.6 years, p=<0.0001), were transplanted more frequently in CR1 (42% vs 24%, p=<0.001), less frequently in advanced disease (10% vs 21%, p=<0.001) and had more often negative cytomegalovirus (CMV) serology (45% vs 31%, p=<0.001).Conditioning regimen was mainly total body irradiation (TBI)-based, 56% versus 50% for TCD-Haplo and UCBT, respectively. For UCBT, patients CSA+steroids was the most commonly used (72%) GVHD prophylaxis. A higher proportion of patients transplanted with TCD-Haplo received ATG in the conditioning regimen (90% vs 74%, p=<0.001).Acute GVHD (grade II-IV) incidence was 16% and 28% (p<0.001) while that of chronic GVHD was 14% and 16%, (p=0.40) for TCD-Haplo and UCBT respectively. Since diagnosis was the most important factor influencing outcome, the analysis was performed separately for patients with ALL and AML.For ALL (n=855) in univariate analysis, the 2-year probability of leukemia-free survival (LFS) was 35% and 43% (p=0.08), for TCD-Haplo and UCBT, respectively. The cumulative incidence (CI) of non-relapse mortality (NRM) was 28% and 29% (p=0.57), and relapse incidence (RI) was 36% and 28% (p=0.01) for TCD-Haplo and UCBT, respectively.According to disease status, for patients in CR1 (n=300), 2-year RI was 21% versus 20%, p=0.75; NRM 30% versus 24%, p=0.59; and 2- year LFS 49% versus 56%, p=0.87; for TCD-Haplo and UCBT, respectively.For patients in CR2 (n=478), 2-year RI was 34% versus 29%, p=0.75; NRM 28% versus 32%, p=0.29; and 2- year LFS 38% versus 39%, p=0.63, for TCD-Haplo and UCBT, respectively. For patients in advanced disease (n=77), 2-year LFS was 5% versus 8%, p=0.08 for TCD-Haplo and UCBT, respectively.For patients with AML (n=478), 2-year probability of LFS was 21% and 58% (p=<0.0001), for TCD-Haplo and UCBT, respectively. CI of NRM was 43% and 19% (p=<0.001), and RI was 36% and 23% (p=0.01) for TCD-Haplo and UCBT, respectively.According to disease status, for patients in CR1 (n=207), 2-year RI was 31% versus 14%, p=0.06; NRM 45% versus 16%, p=0.002; and 2-year LFS 24% versus 69%, p=<0.001; for TCD-Haplo and UCBT, respectively.For patients in CR2 (n=180), 2-year RI was 28% versus 24%, p=0.49; NRM 44% versus 21%, p=0.004; and 2- year LFS 28% versus 55%, p=0.0005, for TCD-Haplo and UCBT, respectively. For patients in advanced disease status (n=91), 2-year LFS was 10% versus 22%, p=0.44 for TCD-Haplo and UCBT, respectively.In multivariate analysis adjusted for differences between the 2 groups, for patients with ALL, no statistically significant differences were observed according to the graft source. Disease status was the only factor associated with better LFS (HR 4.84, p<0.0001). TCD-Haplo was associated with greater risk of RI (HR 1.58, p=0.01).For AML, in multivariate analysis, TCD-Haplo was associated with greater risk of relapse (HR 1.67, p=0.05) and of NRM (HR= 1.94; p=<0.001), and worse LFS (HR 1.94, p=<0.001) when compared to UCBT. Advanced disease at transplantation (HR 2.89, p=<0.001) was the other factor associated with LFS and RI.This retrospective analysis demonstrates that children with ALL have comparable probability of LFS after either UCBT or TCD-Haplo. By contrast, in children with AML, UCBT is associated with lower risk of relapse and NRM than TCD-Haplo, this translating into better LFS. These results may help guide physician choices for transplanting children with acute leukemia. DisclosuresNo relevant conflicts of interest to declare.

Normal RUNX1 and Pathogenic ETV6/RUNX1 Compete Genome-Wide for Chromatin Binding in Pre-B Acute Lymphoblastic Leukemia

Introduction and objectives:The ETV6/RUNX1 fusion protein is a leukemia-initiating transcription factor that interferes with normal RUNX1 transcription factor function. Although RUNX1 can be either a repressor or an activator, ETV6/RUNX1 is mainly associated with transcriptional repression. Both RUNX1 and ETV6/RUNX1 proteins are expressed in pre-B acute lymphoblasts and bind the same DNA binding motif, which is normally recognized by the RUNX1 DNA binding domain, therefore competing for gene regulation with possible adverse transcriptional effects. Here, (we address the question of how) do the abnormal transcription factor ETV6/RUNX1 and the normal RUNX1 interplay at the chromatin level to modulate expression of genes, possibly giving rise to leukemia.Materials and methods:We used chromatin-immunoprecipitation coupled with sequencing (ChIP-seq) both in pre-B leukemic REH cells, which originate from a relapse of an ETV6/RUNX1 leukemic patient and lack the non-translocated allele of ETV6, and in pre-B leukemic Nalm6 cells that are ETV6/RUNX1-negative, to characterize the genomic distribution of ETV6/RUNX1 and RUNX1, as well as transcriptionnally active and inactive chromatin regions (ChIP-seq for methylated H3K4me3 and H3K4me1 histones, and acetylated H3K27achistones). In addition, we ran microarrays experiments to define the transcriptional programme associated with ETV6/RUNX1 and RUNX1 expression in pre-B cell lines. Data were compared to gene expression data from ETV6/RUNX1 pre-B acute lymphoblastic leukemia samples.Results:Through mapping the genome-wide distribution of RUNX1 et ETV6/RUNX1, we identified both common and exclusive DNA sites. We further characterized the DNA binding motifs of RUNX1 and ETV6/RUNX1, which were particularly enriched within particular transposon sequences. ChIP-seq of histone marks identified active and inactive chromatin in correlation with ETV6/RUNX1- and RUNX1-binding. Direct target genes for both transcription factors were defined and used for gene ontology analysis, allowing to uncover specific biological functions associated with ETV6/RUNX1 and RUNX1 binding events. Finally, we show that a fraction of the genes misregulated in pre-B acute lymphoblastic leukemia patients are directly controlled by ETV6/RUNX1 and RUNX1.Conclusion:Our data give new insights on the competition between the fusion protein ETV6/RUNX1 and normal RUNX1 in pre-B acute lymphoblastic leukemia at chromatin level genome-wide.This work was supported by CNRS, University of Rennes 1, University Hospital of Rennes, Ligue Régionale contre le Cancer (sections 22, 35 and 56) (MPA, VG, MBT), Région Bretagne (MBT, VG), Rennes Métropole (MBT), SFR Biosit UMS 3480 (VG, MBT), Société Française de Lutte contre les Cancers et les Leucémies de l’Enfant et de l’Adolescent and the Fédération Enfants et Santé (MBT), Association Laurette Fugain (VG) and the People Programme (Marie Curie Actions) of the European Union Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n°291851 (MBT). DisclosuresNo relevant conflicts of interest to declare.

VACCINATION OF PATIENTS WITH ONCOLOGY DISEASES AGAINST MEASLES AND MUMPS

Abstract. One hundred and seven children (45 girls and 62 boys) in the age of 20 months — 14 years old (mean 9,62±0,37) suffered from acute lymphoblast leukosis and solid tumors in history have been examined in the clinic of Research Institute of children infections of FMBA. The vaccination history was studied in all children and the titers of specific antibodies to measles and mumps viruses as well as immune status were determined. 83,8% and 85,4% of studied children had no protection against measles and mumps respectively or had low titers of antibodies. Immunological examination of these children conducted within 4 months after finishing of therapy revealed absence of immunodeficiency. It gave opportunities to vaccinate or revaccinate these children against mentioned infections. Fifty three children were immunized against measles and 47 — against mumps. Application of live vaccines was safe because majority of vaccinated against measles (81,1%) and mumps (82,9%) children had mild vaccination process. It was established that to increase immunological efficacy of vaccination using of polyoxidony during 5 days before vaccination and 5 days after vaccination is reasonable.