scholarly journals Hhe results of different antibodies application for CD1a expression evaluation in pediatric t-lineage all

2019 ◽  
Vol 17 (4) ◽  
pp. 23-26 ◽  
Author(s):  
A. S. Aksenova ◽  
O. I. Illarionova ◽  
D. V. Litvinov ◽  
S. A. Kashpor ◽  
A. M. Popov
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Lymphoblastic Leukemia ◽  
Antigen Expression ◽  
Expression Data ◽  
Multicolor Flow Cytometry ◽  
Tumor Cell Population ◽  
Multicenter Trials ◽  
Flow Cytometric ◽  
Expression Evaluation

CD1a antigen expression is an important prognostic factor in T-lineage acute lymphoblastic leukemia (T-ALL), thus standardized approach for this antigen expression detection is crucial for multicenter trials. The use of different antibodies in laboratories could lead to wrong decisions for patients management. The aim of the present study was to analyze the results of flow cytometric bone marrow investigation in children with T-ALL using different CD1a-directed antibodies. The bone marrow samples from 31 children (8 girls and 23 boys) with T-ALL aged from 1 to 16 years (median age 7) were studied by multicolor flow cytometry including two different antibodies against CD1a (BL6 и SK9). There were no significant differences in the immunophenotyping results. However, the CD1a-positivity of tumor cell population was visible in the dot plot better when BL6 antibody was used. In addition, two patients with discordant CD1a expression data were founded. Therefore, we antibody BL6 was recommended for routine T-ALL immunophenotyping.

Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndrome: A Retrospective Validation From a Single Centre.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4846-4846
Author(s):  
Pervin Topcuoglu ◽  
Klara Dalva ◽  
Sule Mine Bakanay ◽  
Sinem Civriz Bozdag ◽  
Onder Arslan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndrome ◽  
Antigen Expression ◽  
Pathological Examination ◽  
Light Scatter ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Altered Expression ◽  
Flow Cytometric

Abstract Abstract 4846 Myelodysplastic syndrome (MDS) is heterogeneous clonal hematopoietic stem cell disorder characterized by cytopenia(s) and dysplasia in one or more cell lineage. Though flow cytometry (FCM) is an important diagnostic tool in hematopoietic cell disorders, a prominent immunophenotyping feature in MDS may not be determined. In this study, we retrospectively evaluated flow cytometric features of bone marrow samples diagnosed as MDS with clinical and hematological findings. Patients-Method Between Feb 2004 and March 2009, flow cytometric parameters of 73 patients (M/F: 50/23) with MDS were re-analyzed. Median age was 59 years (17-89 ys). Our general principles are to evaluate quality of bone marrow samples, to determine proportion of the cells and features of their light scatter, and to give percentage of the blast. When detected a finding of dysplasia in the first analysis, the second step includes the determination of the maturation of the cells and the presence of the aberrant antigen expression. Results The samples were interpreted as MDS in % 76.7, MDS-RAEB-1 or RAEB-2 in %16.4, myeloproliferative disorder in %1.4 and non-diagnostic in %6.8 of the cases by flow cytometric examination. We detected variable degrees of hypogranulation in myeloid lineage in %82.2 of the samples by the light scatter features of the cells: 85% of severe and 15% of moderate or mild hypogranulation. The ratio of myeloid and lymphoid was changing from 0.3 to 17.5 (median 2). The decreasing of this ratio (<1) was observed in 19.4% of the samples. We detected altered expression of mature granulocyte. These included decreasing or lack of expression in CD15 45/73 (61.4%), CD13 38/70 (54.3%), CD16 53/67 (79.1%), CD11b 51/71 (71.8%), CD24 44/69 (65.2%), CD10 23/72 (31.9%) and MPO 14/72 (19.4%). Besides, bright expression of CD33 in 53.5% of the samples was observed. CD36 and CD56 in myeloid lineage were co-expressed in about 50 % of the samples. In 80.8 % of the samples dysplasia in erythroid compartment could be evaluated: Expression of CD71 according to glycophorin A (ratio <1) was decreased in 23.7 %. When we made similar analysis in the samples without RAEB-1 and -2 as pathological examination of bone marrow, 13.4 % of the samples could not be evaluated in favor of dysplasia. Of the samples with dysplasia hypogranulation, aberrant antigen expression of myeloid lineage and eryhtroid dysplasia were observed in 92.1%, 34.1% and 31.5%, respectively. In conclusion, FCM events may help to the differantial diagnosis of MDS especially when combining with clinical events. Improving of the analysis by focusing on the blast characteristics may be a standard approach to evaluate for low risk MDS. Disclosures No relevant conflicts of interest to declare.

BCR-ABL fusion protein detection in peripheral blood and bone marrow samples of adult precursor B-cell acute lymphoblastic leukemia patients using the flow cytometric immunobead assay

2012 ◽  
Vol 50 (9) ◽  
Author(s):  
Eirini E. Grigoriou ◽  
Katerina K. Psarra ◽  
Maria K. Garofalaki ◽  
Eirini C. Tziotziou ◽  
Chryssa A. Papasteriades
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Fusion Protein ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Protein Detection ◽  
Flow Cytometric ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Polymerase Chain

AbstractThe ability to detect theWe used a new flow cytometric immunobead assay for BCR-ABL fusion protein detection in peripheral blood and/or bone marrow samples from 38 adult pB-ALL patients and the results were compared with polymerase chain reaction (PCR) detection of BCR-ABL transcript.The fusion protein was detected in peripheral blood and bone marrow samples from seven of the 38 (18%) patients, and results for both the p190 and p210 were confirmed by PCR. One case, which was positive by cytogenetics and fluorescence in situ hybridization (FISH), was negative by PCR but positive by flow cytometry. Another case, which was positive by PCR and negative by flow cytometry, was from a patient on steroid treatment.The cytometric immunobead assay for BCR-ABL fusion protein detection was found to be suitable for the investigation of pB-ALL patients. This assay is reliable, rapid and simple to use for peripheral blood and bone marrow samples.

scholarly journals Immunophenotypic Characterization of Human Bone Marrow Mast Cells. A Flow Cytometric Study of Normal and Pathological Bone Marrow Samples

1998 ◽  
Vol 16 (3) ◽  
pp. 151-159 ◽  
Author(s):  
Luis Escribano ◽  
Alberto Orfao ◽  
Jesús Villarrubia ◽  
Beatriz Díaz-Agustín ◽  
Carlos Cerveró ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mast Cells ◽  
Antigen Expression ◽  
Hematologic Malignancies ◽  
Healthy Controls ◽  
Flow Cytometric ◽  
Hla Dr ◽  
Flow Cytometric Study

The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC) from healthy controls and patients with hematologic malignancies (HM) based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogenous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p= 0.08). Three patterns of antigen expression were detected: (1) markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and FcεRI), (2) antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138), and (3) markers that were positive in a variable proportion of cases – CD11b (50%), CD11c (77%), CD13 (40%), CD18 (20%), CD22 (68%), CD35 (27%), CD40 (67%), CD54 (88%) and CD61 (40%). In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes.In summary, our results show that BMMC from both healthy controls and HM patients display a relatively heterogenous immunophenotype. Interestingly, we have observed clear differences between the immunophenotype of BMMC and MC from other tissues. This could be due either to the heterogeneity of human MC according to their tissue localization or to the sensitivity of the method used for antigen detection.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

scholarly journals The influence of a dosage regimen of dexamethasone on detection of normal B-cell precursors in the bone marrow of children with BCP-ALL at the end of induction therapy

2020 ◽  
Vol 19 (1) ◽  
pp. 53-57
Author(s):  
E. V. Mikhailova ◽  
T. Yu. Verzhbitskaya ◽  
J. V. Roumiantseva ◽  
O. I. Illarionova ◽  
A. A. Semchenkova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Induction Therapy ◽  
Therapy Group ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Intermittent Therapy ◽  
Continuous Therapy ◽  
B Cell Precursors

Minimal residual disease (MRD) monitoring by flow cytometry at the end of induction therapy is one of the key ways of a prognosis assessment in patients with acute lymphoblastic leukemia (ALL). In B-cell precursor ALL (BCP–ALL), this method of MRD detection is complicated due to the immunophenotypic similarity between leukemic cells and normal B-cell precursors (BCPs). A decrease in intensity of induction therapy can lead to a more frequent appearance of normal BCPs in the bone marrow, which significantly complicates the MRD monitoring. Aim: to assess the incidence of normal BCPs in bone marrow on the 36th day of induction therapy with two different regimens of glucocorticoid (GC) administration according to ALL-MB 2015 protocol. This study was approved by the Independent Ethical Committee and the Academic Council of Dmitriy Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology Ministry of Healthcare of Russian Federation. The study included 220 patients with BCP-ALL who were randomized to two types of GC-based induction therapy: a continuous administration of dexamethasone (n = 139) and an intermittent regimen with a 1-week dexamethasone therapy stop (n = 81). On the 36th day of induction therapy, MRD and normal BCPs were quantified in bone marrow samples by flow cytometry. On the 36th day of treatment, 43.2% of BCP(+) samples were established in the intermittent-therapy group, and 27.3% in the continuous-therapy group (p = 0.016). Comparison of the BCP level in BCP(+) samples revealed the more equitable distribution of BCPs at different developmental stages in the intermittent-therapy group, meanwhile mainly the immature BCPs in a quantity of less than 0.01% were found in the continuous-therapy group. Reduced-intensity induction therapy for patients with BCP-ALL leads to a noticeable increase of normal BCPs in bone marrow at the end of this treatment stage. A higher rate of BCP(+) bone marrow samples hinder the MRD detection due to the immunophenotypic similarity of BCPs and leukemic cells.

Monitoring of T-cell acute lymphoblastic leukemia by flow cytometry

Open Medicine ◽  
2010 ◽  
Vol 5 (6) ◽  
pp. 651-658
Author(s):  
Miglė Janeliūnienė ◽  
Rėda Matuzevičienė ◽  
Laimonas Griškevičius ◽  
Zita Kučinskienė
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric ◽  
Analysis Strategy ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Positive Results ◽  
Color Marker

AbstractMinimal residual disease (MRD) predicts the outcome of acute lymphoblastic leukemia (ALL). Flow cytometry (FC) is one of the most sensitive and most applicable methods for MRD diagnostics, but there is still no agreement on the “gold standard” of the method. We tried to optimize flow cytometric MRD detection in T-ALL. Fourteen adults and 11 children with T-ALL and 12 normal bone marrow (BM) donors were enrolled in the study. We found that the most common phenotypic aberrations in T-ALL were TdT and CD99 coexpression on T-cells in BM. Therefore for MRD detection we developed a limited four-color marker panel (TdT/CD7/cCD3/CD19 and CD99/CD7/cCD3/CD2) and a standard analysis strategy. This assay was evaluated on BM of healthy controls. Less than 0.01% TdT+ or CD99 bright T-cells were found in normal BM. MRD was detected in 9 adult patients and 1 child at different time-points of treatment. The average TdT and CD99 mean fluorescence intensity (MFI) value of residual blasts fluctuated during therapy, but it still remained higher than MFI of normal T-cells. Our established MRD detection method differentiated leukemic lymphoblasts with sensitivity in the range of 0.01% and did not give any false positive results in normal BM.

scholarly journals Myelomonocytic antigen positive multiple myeloma

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 763-769 ◽  
Author(s):  
TM Grogan ◽  
BG Durie ◽  
CM Spier ◽  
L Richter ◽  
E Vela
Keyword(s):  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Antigen Expression ◽  
Survival Duration ◽  
Double Labelling ◽  
Myeloma Cells ◽  
Myeloid Antigens ◽  
A Cell ◽  
Multilineage Potential ◽  
Leukemia Antigen

Abstract In a four year span, between 1983 and 1987, 215 bone marrow and cell culture samples from 125 myeloma patients were immunotyped and coexpression of myelomonocytic and plasma cell antigens occurred in 16 (13%). We employed both immunohistochemical and flow cytometry methods including coplots and double labelling. Three types of myeloma cases were found: (1) those with isolated myeloid antigen coexpression, usually Leu M1 or esterase (BE, CE) positive (11 cases); (2) those with multiple myeloid antigens (Leu M1, M3, M5, MY7, BE, CE) (four cases); and (3) one case beginning as 1 and ending as 2. Isolated myeloid antigen expression was generally associated with typical features of myeloma with survival close to the anticipated median (33 months), while multiple myeloid antigen expression was associated with more aggressive disease and shorter survival duration (median survival 16 months). The latter subgroup also had other poor prognostic factors including high labelling index and common acute lymphoblastic leukemia antigen (CALLA) positivity. Other features found overall were frequent abnormal karyotypes (seven of 12 abnormal) and coexpressed IgA (eight of 16); all IgA+ cases also coexpressed Leu M1. We conclude that there is an unusual and unexpected predilection for coexpression of myelomonocytic antigens in myeloma cells. The reasons are not immediately obvious. Whether the coexpression indicates that myeloma cells truly have latent multilineage potential or just aberrantly coexpress other hematopoietic antigens as a manifestation of malignancy remains to be explained. However, a cell line established from the bone marrow of one patient is a valuable scientific tool allowing detailed analysis of these questions.

scholarly journals CD2 antigen expression on leukemic cells as a predictor of event-free survival after chemotherapy for T-lineage acute lymphoblastic leukemia: a Children's Cancer Group study

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4288-4295 ◽  
Author(s):  
FM Uckun ◽  
PG Steinherz ◽  
H Sather ◽  
M Trigg ◽  
D Arthur ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Lymphoblastic Leukemia ◽  
Antigen Expression ◽  
Leukemic Cells ◽  
Event Free Survival ◽  
Free Survival ◽  
Cancer Group ◽  
Standard Risk

Abstract We examined the prognostic impact of CD2 antigen expression for 651 patients with T-lineage acute lymphoblastic leukemia (ALL), who were enrolled in front-line Childrens Cancer Group treatment studies between 1983 and 1994. There was a statistically significant correlation between the CD2 antigen positive leukemic cell content of bone marrow and probability of remaining in bone marrow remission, as well as overall event-free survival (EFS) (P = .0003 and P = .002, log-rank tests for linear trend). When compared with patients with the highest CD2 expression level (> 75% positivity), the life table relative event rate (RER) was 1.22 for patients with intermediate range CD2 expression level (30% to 75% positivity) and 1.81 for “CD2-negative” patients (< 30% positivity). At 6 years postdiagnosis, the EFS estimates for the three CD2 expression groups (low positivity to high positivity) were 52.8%, 65.5%, and 71.9%, respectively. CD2 expression remained a significant predictor of EFS after adjustment for the effects of other covariates by multivariate regression, with a RER of 1.47 for CD2- negative patients (P = .04). Analysis of T-lineage ALL patients shows a significant separation in EFS after adjustment for the National Cancer Institute (NCI) age and white blood cell (WBC) criteria for standard and high-risk ALL (P = .002, RER = 1.67). The determination of CD2 expression on leukemic cells helped identify patients with the better and poorer prognoses in both of these risk group subsets. For standard risk T-lineage ALL, CD2-negative patients had a worse outcome (P = .0007, RER = 2.92) with an estimated 5-year EFS of 55.9% as compared with 78.3% for the CD2-positive patients. Thus, CD2 negativity in standard risk T-lineage ALL identified a group of patients who had a worse outcome than high-risk T-lineage ALL patients who were CD2 positive. The percentage of CD2 antigen positive leukemic cells from T- lineage ALL patients is a powerful predictor of EFS after chemotherapy. This prognostic relationship is the first instance in which a biological marker in T-lineage ALL has been unequivocally linked to treatment outcome.

Augmentation of Sensitivity of FLT3/ITD Assay Allows Detection of Minimal Residual Disease In Stem Cell Transplant Recipients-Correlation with Flow Cytometric MRD Assessment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1717-1717
Author(s):  
Maya Danielle Hughes ◽  
Rong Zeng ◽  
Kristen L. Miller ◽  
Soheil Meshinchi
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Flow Cytometric ◽  
Detection Assay ◽  
Minimal Residual

Abstract Abstract 1717 FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutation that is associated with therapy resistance in acute myeloid leukemia (AML). Early data demonstrated low sensitivity for this assay, thus limiting its utility to the evaluation of diagnostic specimens, and precluding its utility in remission samples. We inquired whether the standard FLT3/ITD assay can be modified to enable its utility to detect presence of residual disease in remission specimens. Enhanced FLT3/ITD assay sensitivity was accomplished by altering annealing temperature, increasing the number of cycles as well as amount and concentration of the product that was subjected to capillary electropheresis. To assess the sensitivity of the enhanced assay, FLT3/ITD positive cells M4V11 were serially diluted in a population of ITD negative cells (HL60). The concentration of M4V11 cells in each sample ranged from 10% to 0.0001%. PCR product was subjected to capillary electropheresis and the appropriate region of the electropherogram was examined for the presence of the appropriate mutant product length. Appropriate FLT3/ITD signal was detected in dilutions down to 0.01%, validating our ability to detect extremely low levels of FLT3/ITD. We subsequently examined the remission marrows from patients with a history of FLT3/ITD who had undergone stem cell transplantation. Available bone marrow specimens (N = 51) from patients who underwent stem cell transplantation for FLT3/ITD-positive AML were analyzed and the result was correlated with the available standard PCR as well as the available MRD assessment by muti-dimensional flow cytometry; samples negative for FLT3/ITD by standard assay (N=11) were then subjected to the enhanced PCR methodology. Available ITD length for each patient was used for examination of the appropriate region of the electropherogram in each case. Of the available 51 bone marrow specimens analyzed, 23 specimens had FLT3/ITD detectable by standard PCR protocol. Using our modified PCR method and capillary electrophoresis, an additional 13 specimens had identifiable FLT3/ITD. In 6/11 patients, where initial FLT3/ITD was negative by standard methodology, enhanced assay identified FLT3/ITD signal. In each case, detection of FLT3/ITD by the enhanced assay was followed by morphologic or immunophenotypic emergence of disease, prompting therapeutic intervention. We further evaluated the ability to detect FLT3/ITD in patients with minimal residual disease by flow cytometry. 33 of the bone marrow specimens analyzed had a less than 5% abnormal blast population as detectable via flow cytometry. Among these samples, 7 had FLT3/ITD detectable using standard detection techniques. An additional 11 samples had detectable FLT3/ITD when our modified protocol was employed. Of the specimens that had less than 1% abnormal blast population as detectable via flow cytometry (N = 27), 4 had FLT3/ITD detectable using the standard detection assay; when our modified protocol was employed, an additional 6 samples had detectable FLT3/ITD. 17 bone marrow specimens had no abnormal blast cells detectable via flow cytometry; of these samples 1 had detectable FLT3/ITD using the standard detection assay, while an additional 3 had detectable FLT3/ITD using our modified assay. In four patients, FLT3/ITD was detected in bone marrow specimens found to have flow cytometric MRD of 0% (N=2), 0.1% (N=1) and 0.4% (N=1). In two patients with no detectable disease by MDF, both had emergence of morphologic (60% blast) or immunophenotypic disease by MDF (1.1%) within 4–6 weeks of detection of FLT3/ITD by enhanced assay. In this study, we demonstrate that simple modifications to the FLT3/ITD genotyping assay significantly increases its sensitivity and provides a highly sensitive and very specific assay for identifying this disease associated mutation in remission specimens. The enhanced assay can be incorporated into the standard evaluation of remission status for patients with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.

Dnmt3a Deletion and FLT3-ITD Cooperate in a Mouse Model of T-Lymphoblastic Leukemia (T-ALL).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2428-2428
Author(s):  
Liubin Yang ◽  
Min Luo ◽  
Mira Jeong ◽  
Choladda V. Curry ◽  
Grant Anthony Challen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Leukemic Transformation ◽  
Aberrant Dna Methylation

Abstract Abstract 2428 Aberrant DNA methylation repeatedly has been implicated in cancer development. DNA methyltransferase (DNMT) 3A, which mediates de novo DNA methylation, was found to be mutated in 20% of patients with acute myeloid leukemia and 10% of patients with myelodysplastic syndrome. Recently, mutations associated with myeloid malignancies such as DNMT3A and FLT3 have also been uncovered in patients with early T-cell precursor lymphoblastic leukemia (ETP-ALL) (Neumann et al., 2012; Van Vlierberghe et al., 2011; Zaremba et al., 2012). ETP-ALL is a type of very high-risk ALL associated with myeloid/stem cell gene expression signature and myeloid markers. We have demonstrated that Dnmt3a deletion in mouse causes increased self-renewal of hematopoietic stem cells and an impairment of differentiation (Challen et al., 2011). Dnmt3a loss also produces aberrant methylation associated with oncogenes and tumor suppressor genes. Yet, whether aberrant DNA methylation can drive leukemia remains unknown. As Dnmt3a deletion alone was insufficient for malignancy, secondary mutations are likely necessary for leukemic transformation. Because FLT3 internal tandem duplication (ITD) frequently co-exist with DNMT3A mutations in acute leukemias, we hypothesized that Dnmt3a-loss may cooperate with FLT3-ITD to promote leukemic transformation; and we established a mouse model to test this. Deletion of conditional Dnmt3a with Mx1-cre was induced by injections of pIpC. Subsequently, bone marrow from Dnmt3a-deleted (Dnmt3aKO) donor mice was transduced with MSCV-FLT3-ITD-GFP retrovirus or MSCV-GFP control and transplanted into lethally irradiated recipients. The mice were monitored monthly for development of malignancies by complete blood count and peripheral blood analysis by flow cytometry and followed for disease latency. Moribund mice were sacrificed and analyzed with peripheral blood smears, histology, and immunophenotyping. Dnmt3a deletion with overexpression of FLT3-ITD caused rapid onset T-ALL in 6/8 mice (n=6) with a median latency of 78 days compared to 121 days in WT mice (n=4) overexpressing FLT3-ITD (p&lt;0.0001 Log-rank Mantel-Cox Test) (See figure). Mice from both groups exhibited leukocytosis, splenomegaly, and thymomegaly with high GFP expression detected by FACS. Even after we transduced bone marrow cells enriched for myeloid progenitor and stem cells, Dnmt3a deletion again accelerated T-ALL with median survival of 89 days (n=9) versus 110 days in WT-FLT3-ITD (n=10) mice. T-ALL was observed in 2/4 WT-FLT3-ITD mice and 5/6 Dnmt3aKO-FLT3-ITD mice analyzed (p&lt;0.0001 Log-rank Mantel-Cox Test). By flow cytometry, two distinct types of T-ALL were observed in the bone marrow of Dnmt3a deleted leukemic mice: one was characterized by a double positive population (DP) of CD4+CD8+ lympoblasts (1/6) and another early immature T-cell-like type of CD4-CD8-CD44+CD25-CD11bloCD117+ lymphoblasts (4/6). Gene expression analysis by RT-PCR in the early immature T-ALL showed downregulation of Notch-pathway genes (such as Notch1, Notch 3, Deltex, Hes1) and upregulation of stem cell-associated genes Lyl1 and Scl1, suggesting an ETP-like T-ALL. The ETP-like ALL phenotype has not been seen in WT mice overexpressing FLT3-ITD. The opposite gene expression pattern was seen in the DP population with upregulation of Notch-pathway genes. Furthermore, the DP leukemia was transplantable to secondary recipients within 2 weeks. Whether ETP-like ALL can be transplanted is still under investigation. We are also currently studying the changes in global CpG methylation among the leukemias that have Dnmt3a loss, FLT3-ITD overexpression, and control and also anticipate data from transcriptome analysis by RNA-Seq. These data suggest that stem or progenitor bone marrow cells primed by early loss of Dnmt3a are transformed into DP T-ALL and ETP-like ALL fueled by the overexpression of the oncogene FLT3-ITD. The ETP-like ALL phenotype has not been seen previously in WT mice overexpressing FLT3-ITD, suggesting that Dnmt3a ablation is required. The Dnmt3a-deleted-FLT3-ITD mice with T-ALL is, to our knowledge, the first animal model of human immature T-cell leukemia. This model can enhance our understanding of the pathogenesis of ETP-like ALL with respect to aberrant DNA methylation and will serve as a powerful tool to test novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

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