Establishment and Characterization of A New Leukemia Mouse Model Induced by MLL/AF4 Fusion Protein.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3573-3573
Author(s):  
Hayato Tamai ◽  
Koichi Miyake ◽  
Noriko Miyake ◽  
Hiroki Yamaguchi ◽  
Kazuo Dan ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Mixed Lineage Leukemia ◽  
Lymphoid Cells ◽  
Long Latency ◽  
Infant Leukemia

Abstract Abstract 3573 Poster Board III-510 Background MLL/AF4 fusion is the most common abnormality in infant leukemia and is associated with a poor prognosis. Although further study and treatment should be groped for acute lymphoblastic leukemia (ALL) with MLL/AF4, the lack of a proper animal model has impeded understanding of the molecular mechanism of leukemia associated with the MLL/AF4 fusion gene. Previous mouse models suggest that the tumorigenesis capacity of MLL/AF4 alone is insufficient for development mixed-lineage leukemia. MLL/AF4 is capable of inducing lymphoid malignancy with long latency, which is very different from aggressive pro-B ALL in humans. Recently, we have reported that enhancement of S100A6 (calcyclin) expression played an important role in MLL/AF4-leukemogenesis using murine 32Dc cell line. In this study, we tried to make improved MLL/AF4 transgenic (Tg) mice which reflect human leukemogenicity and analyzed molecular mechanism of MLL/AF4 fusion gene. Methods To get high and sustained expression of MLL/AF4 in hematological lineage, we used MSCV promoter to express MLL/AF4 fusion gene. After MLL/AF4 expression plasmids were injected into fertilized eggs of mice (C57BL/6), the manipulated embryos were transferred into the oviducts of pseudopregnant females to make MLL/AF4 Tg mice. Finally we characterized MLL/AF4 Tg mice and analyzed molecular mechanism of MLL/AF4-leukemogenesis. Results Our new established MLL/AF4 Tg mice had lymphoma earlier than previous MLL/AF4 knock-in mice (median 12M vs 17M). Infiltration of lymphoid cells was located in liver, lung, kidney, and spleen and they consist of pro B-cell (CD45R+CD43+CD19-) lineage. Leukemic change developed two months (median 14M) after appearance of lymphoma. More than 30% of abnormal lymphocytes and severe anemia (RBC: 276×104 ml, Hb: 4.9 g/ml) were found in peripheral blood in MLL/AF4 Tg mice. Western blot analysis showed that up-regulation of HoxA9 and S100A6 was detected in MLL/AF4 Tg mouse compared to wild type C57BL/6. Conclusion We succeeded in establishment of improved more aggressive MLL/AF4 Tg mice which reflect MLL/AF4 leukemogenicity of humans. Appearance of lymphoma in our MLL/AF4 Tg mice needed short er latency than those of the previous knock-in mice. Enhancement of HoxA9 and S100A6 expression was involved in MLL/AF4-associated leukemogenesis. We believe that this new MLL/AF4 Tg mouse is useful for study of the molecular mechanisms of MLL/AF4 leukemogenesis and development of new therapy for mixed-lineage leukemia. Disclosures: No relevant conflicts of interest to declare.

Transforming and Tumorogenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosin-Kinase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4683-4683
Author(s):  
Álvaro Cuesta-Domínguez ◽  
Mara Ortega ◽  
Cristina Ormazabal ◽  
Matilde Santos-Roncero ◽  
Marta Galán-Díez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Nude Mice ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Chimeric Protein ◽  
Tyrosine Kinase Domain ◽  
Molecular Response

Abstract Abstract 4683 Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors. Disclosures: No relevant conflicts of interest to declare.

Targeting Kinase-activating Genetic Lesions to Improve Therapy of Pediatric Acute Lymphoblastic Leukemia

2018 ◽  
Vol 25 (24) ◽  
pp. 2811-2825 ◽  
Author(s):  
Raffaella Franca ◽  
Natasa K. Kuzelicki ◽  
Claudio Sorio ◽  
Eleonora Toffoletti ◽  
Oksana Montecchini ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Fusion Gene ◽  
Hematologic Malignancy ◽  
Lymphoblastic Leukemia ◽  
Point Of View ◽  
Lymphoid Cells ◽  
Gene Encoding ◽  
Pediatric All ◽  
Blast Cells ◽  
Tk Inhibitors

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children, characterized by an abnormal proliferation of immature lymphoid cells. Thanks to risk-adapted combination chemotherapy treatments currently used, survival at 5 years has reached 90%. ALL is a heterogeneous disease from a genetic point of view: patients’ lymphoblasts may harbor in fact several chromosomal alterations, some of which have prognostic and therapeutic value. Of particular importance is the translocation t(9;22)(q34;q11.2) that leads to the formation of the BCR-ABL1 fusion gene, encoding a constitutively active chimeric tyrosine kinase (TK): BCR-ABL1 that is present in ~3% of pediatric ALL patients with B-immunophenotype and is associated with a poor outcome. This type of ALL is potentially treatable with specific TK inhibitors, such as imatinib. Recent studies have demonstrated the existence of a subset of BCR-ABL1 like leukemias (~10-15% of Bimmunophenotype ALL), whose blast cells have a gene expression profile similar to that of BCR-ABL1 despite the absence of t(9;22)(q34;q11.2). The precise pathogenesis of BCR-ABL1 like ALL is still to be defined, but they are mainly characterized by the activation of constitutive signal transduction pathways due to chimeric TKs different from BCR-ABL1. BCR-ABL1 like ALL patients represent a group with unfavorable outcome and are not identified by current risk criteria. In this review, we will discuss the design of targeted therapy for patients with BCR-ABL1 like ALL, which could consider TK inhibitors, and discuss innovative approaches suitable to identify the presence of patient’s specific chimeric TK fusion genes, such as targeted locus amplification or proteomic biosensors.

scholarly journals Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 49-49
Author(s):  
Qiuling Chen ◽  
Yuelong Jiang ◽  
Qinwei Chen ◽  
Long Liu ◽  
Bing Xu
Keyword(s):  
B Cell ◽  
Solid Tumors ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Lymphocytic Leukemia ◽  
Advanced Lung Cancer ◽  
Antitumor Effects

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status. Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms. In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3. In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

scholarly journals Identification and Characterization of Novel Fusion Genes with Potential Clinical Applications in Mexican Children with Acute Lymphoblastic Leukemia

2019 ◽  
Vol 20 (10) ◽  
pp. 2394 ◽  
Author(s):  
Minerva Mata-Rocha ◽  
Angelica Rangel-López ◽  
Elva Jiménez-Hernández ◽  
Blanca Angélica Morales-Castillo ◽  
Carolina González-Torres ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Fusion Genes ◽  
Cancer Etiology ◽  
Mexican Children ◽  
Genes Encoding ◽  
Identification And Characterization ◽  
Current Risk

Acute lymphoblastic leukemia is the most common type of childhood cancer worldwide. Mexico City has one of the highest incidences and mortality rates of this cancer. It has previously been recognized that chromosomal translocations are important in cancer etiology. Specific fusion genes have been considered as important treatment targets in childhood acute lymphoblastic leukemia (ALL). The present research aimed at the identification and characterization of novel fusion genes with potential clinical implications in Mexican children with acute lymphoblastic leukemia. The RNA-sequencing approach was used. Four fusion genes not previously reported were identified: CREBBP-SRGAP2B, DNAH14-IKZF1, ETV6-SNUPN, ETV6-NUFIP1. Although a fusion gene is not sufficient to cause leukemia, it could be involved in the pathogenesis of the disease. Notably, these new translocations were found in genes encoding for hematopoietic transcription factors which are known to play an important role in leukemogenesis and disease prognosis such as IKZF1, CREBBP, and ETV6. In addition, they may have an impact on the prognosis of Mexican pediatric patients with ALL, with the potential to be included in the current risk stratification schemes or used as therapeutic targets.

Short Time Latency in Infant Leukemia with ETV6/RUNX1 fusion Gene

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4882-4882
Author(s):  
Mariana Emerenciano ◽  
Silvia Bungaro ◽  
Giovanni Cazzaniga ◽  
Maria Dolores Fonseca Dorea ◽  
Virginia Maria Coser ◽  
...  
Keyword(s):  
Epidemiological Study ◽  
Copy Number ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Neutral Loss ◽  
Control Group ◽  
Rt Pcr ◽  
Genomic Deletions ◽  
Age Related ◽  
Infant Leukemia

Abstract Acute lymphoblastic leukemia (ALL) in infants is characterized by a high frequency of MLL gene rearrangements. By contrast, the ETV6/RUNX1 (TEL/AML1) fusion gene is usually detected in children older than 2 years. In a series of Brazilian infant leukemia cases collected in an epidemiological study, a screening was performed to identify fusion genes found in ALL, we have identified four cases harboring ETV6/RUNX1 in blast cells of infants with 2, 3, 5 and 7 months-old, and common-ALL. To underlie additional genomic hits required to accelerate the emergence of a frank leukemia in these c-ALL cases, we applied the SNP-based genomic copy number analysis, comparing t(12;21) infants with other children with c-ALL in older age-range groups. The presence of ETV6/RUNX1 fusion gene was demonstrated by reverse transcription polymerase chain reaction (RT-PCR) and confirmed by fluorescence in situ hybridization (FISH). All cases were negative for MLL rearrangements, as assessed both by RT-PCR and FISH analyses. As control group (non-leukemic children), we selected 5 samples from children younger than 2 years-old already included in a previous epidemiological study. Infant cases described here present high WBC count (on average 131 × 109/L), and adverse prognosis: two of them relapsed, and one patient only (relapsed) is still alive four years after diagnosis. Recurrent deletions, including 9p21.3 (CDKN2A/2B and MTAP), 11p13 (CD44), 12p13.2 (ETV6) and regions affected by copy number neutral loss of heterozigosity (LOH) were observed. The frequency of genomic deletions and amplifications detected by 100K SNP arrays varied significantly between age-related c-ALL subgroups. The infant leukemia group showed the lower number of chromosome imbalances, whereas the mean number of deletions was similar in the <2 years and 2–10 years age groups, consistent with the observation that the number of gains was largely increasing in older children. It has been postulated that infant leukemia might be caused by transplacental exposures during pregnancy. Then, with these unusual results we have reviewed the data of their mothers’ interviews. Mothers of infants were effectively exposed to a non-steroidal anti-inflammatory drug, and other chemical substances. It may be hypothesize that the pre-leukemic phase in these age-related subgroups of ETV6/RUNX1 positive patients may be evolved with different latencies due to the occurrence of a combination of genetic and environmental factors.

Distinct Roles for the NF-κB Pathway In Myeloid and Lymphoid Transformation and Leukemogenesis by BCR-ABL.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1225-1225
Author(s):  
Mo-Ying Hsieh ◽  
Richard A. Van Etten
Keyword(s):  
Stem Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Myeloproliferative Neoplasm ◽  
Dominant Negative ◽  
Lymphoid Cells ◽  
Leukemic Cells ◽  
Mutant Form ◽  
B Lymphoid

Abstract Abstract 1225 The BCR-ABL tyrosine kinase, product of the t(9;22) Ph chromosome, activates multiple signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have shown that NF-κB is activated in BCR-ABL-expressing cell lines and contributes to transformation of primary B-lymphoid cells by BCR-ABL (Reuther et al., Genes Dev. 1998;12:968), but the mechanism of activation has not been defined (Kirchner et al., Exp. Hematol. 2003;31:504), and importance of NF-kB to myeloid and lymphoid leukemogenesis by BCR-ABL is unknown. To interrogate the role of NF-κB in BCR-ABL-mediated transformation, we utilized a super-repressor mutant form of IκBα (IκBαSR), which has been used to block NF-κB nuclear localization and transactivation by constitutively sequestering NF-κB in the cytoplasm. Using retrovirus co-expressing BCR-ABL and IκBαSR, we found that IκBαSR blocked nuclear p65/RelA expression and inhibited the IL-3 independent growth of Ba/F3 cells and primary B-lymphoid cells transformed by BCR-ABL. The effect of NF-κB inhibition was primarily on proliferation rather than on cell survival, as there was no increase in apoptosis in cells expressing IκBαSR. When primary bone marrow cells were transduced and transplanted under conditions favoring induction of B-ALL or CML-like myeloproliferative neoplasm in recipient mice, co-expression of IκBαSR significantly attenuated disease development and prolonged survival of diseased mice. Molecular analysis of these leukemias demonstrated that NF-κB inhibition decreased the frequency of leukemia-initiating (“stem”) cells in the CML model, but not in the B-ALL model, and was associated with decreased expression of c-Myc, an NF-κB target. To clarify the mechanism of activation of NF-κB in BCR-ABL-expressing cells, we targeted two upstream kinases that negatively regulate IκBα, IKKα/IKK1 or IKKβ/IKK2. To accomplish this, we engineered retroviruses co-expressing BCR-ABL and kinase-inactive, dominant-negative mutants of IKK1 (IKK1KM) or IKK2 (IKK2KM). Co-expression of either IKK mutant inhibited both B-lymphoid transformation and leukemogenesis by BCR-ABL, as well as induction of CML-like MPN, with IKK1 inhibition more effective than IKK2. Together, these results demonstrate that NF-κB is activated in part through the canonical IKK pathway in BCR-ABL-expressing leukemia cells, and that NF-κB signaling plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL. In CML, NF-κB may play a role for in generation and/or maintenance of leukemic stem cells. These results validate IKKs as targets for therapy in Ph+ leukemias, and motivate the evaluation of small molecule IKK inhibitors in these diseases. Disclosures: No relevant conflicts of interest to declare.

The Pro-Inflammatory IL23/IL23R/IL17 Axis Is Active in IL23R-Expressing Circulating CLL Cells in Patients with Poor Prognosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3889-3889
Author(s):  
Fortunato Morabito ◽  
Giovanna Cutrona ◽  
Anna Grazia Recchia ◽  
Sonia Fabris ◽  
Serena Matis ◽  
...  
Keyword(s):  
Lymph Node ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Lymphoid Cells ◽  
Fish Analysis ◽  
Number Of Patients ◽  
Significant Difference

Abstract Abstract 3889 Inflammatory cytokines play a biological role in the pathogenesis of Chronic lymphocytic leukemia (CLL). IL23 is a pro-inflammatory cytokine involved in T-cell responses and in tissue remodeling. It has been shown that the IL23 receptor (IL23R) is up-regulated in primary acute lymphoblastic leukemia (ALL) cells, and that IL23 inhibits ALL cell growth. Nevertheless, the anti-tumor function of IL23 still remains controversial. The role of the IL23R/IL23 axis in CLL has not been investigated so far. Herein we evaluated the expression pattern of IL23R/IL23 axis and its correlation with progression free survival (PFS) in CLL patients. A total of 233 newly diagnosed Binet stage A CLL cases from Italian institutions (clinicaltrials.gov NCT00917540) were studied for IL23R expression by flow-cytometry (FC) (median percentage IL23R expression=22.7, range 1.2–91.1). The median follow-up was 23 months (range 1–47). PFS information was obtained in 203 patients. Using the median value of 23% of IL23R as threshold, 8/102 IL23Rneg and 23/101 IL23Rpos CLL cases progressed with therapy requirement. The 2-year PFS probability of IL23Rneg patients was 89.7% as compared to 80.7% of IL23Rpos cases [χ2 7.7, P=.006; HR=3.0, 95%CI (1.3–6.6)]. Cases were then stratified according to IL23R positivity [IL23Rneg (102 cases) versus IL23Rpos (101 cases)]. No significant difference in terms of CD38 and ZAP-70 positive cases was observed, however, the IGVH mutational status could distinguish the two groups: IGHV-mutated in 92 (78.6%) of IL23Rneg vs 70 (61.9%) IL23Rpos and IGHV-unmutated in 25 (21.4%) vs 43 (38.1%), p=.006]. FISH analysis showed that IL23Rneg and IL23Rpos cases carrying 13q14.3 were respectively 53 (51.4%) and 44 (42.7%), while the number of patients with trisomy 12 were 8 and 10 respectively in cases with low and high IL23R expression. Deletion of 11q was detected in 3.9% (4/103) of IL23Rneg and in 8.7% (9/103) of IL23Rpos cases. Only 3 cases with 17p deletion were seen in this cohort of early CLL patients and all belonged to the IL23Rpos group. Overall, no significant differences in the incidence of the major genetic lesions were observed between the two groups. Il23R expression still remained independently associated with PFS also in multivariate analysis. In situ expression analysis of IL23R and of its ligand IL23 was then performed by immunohistochemistry (IHC) in 16 CLL samples [10 lymph node (LN) and 6 bone marrow (BM) biopsies] collected on diagnosis and in 8 control biopsies (4 lymph nodes with reactive follicular hyperplasia and 4 normal BM biopsies). IL23R was variably expressed in CLL and significantly expressed in the neoplastic clones of 9 (6 lymph nodes and 3 BM biopsies) of the 16 cases tested; IL23R was diffusely present along the membrane and cytoplasm of neoplastic cells effacing the lymph node or BM architecture (Fig. 1, upper-left). In CLL cases with low IL23R expression, IL23R was detected in few scattered lymphoid cells intermingling with neoplastic lymphocytes (Fig. 1, upper-right). IL23 was also detected, with a variable staining intensity (Fig. 1, middle-left), paralleling in part that of IL23R. Double-marker analysis confirmed the concomitant expression of IL23 and IL23R in CLL neoplastic infiltrates highlighting the co-localization of the two markers (Fig.1 middle-right) and suggesting the possibility of an autocrine IL23/IL23R loop in CLL clones. We speculated that the microenvironment of CLL cases rich in IL23R and IL23 could be enriched in IL17-producing cells. The IHC expression of IL17 in CLL cases with low or high IL23R and IL23 expression showed that CLL cases rich in IL23Rpos cells, also characterized by high IL23 expression, displayed significantly higher numbers of IL17pos infiltrating cells (Fig. 1 bottom-left), as compared with CLL cases with no or low expression of IL23R or IL23 (Fig. 1 bottom-right). In conclusion, our study shows that high IL23R expression predicts a worse PFS. Furthermore, we linked this picture with, the in situ engendering of a clone-related microenvironment characterized by the preponderancy of pro-inflammatory signals such as those of the IL23/IL23R/IL17 axis, and its correlates in the peripheral blood (i.e. IL23R expression on circulating CLL cells), may endorse its strong prognostic significance. This analysis prompts further investigation into the specific function of the IL23/IL23R/IL17 axis and its targets in the context of CLL. Figure 1. Figure 1. Disclosures: No relevant conflicts of interest to declare.

Detection of Five Common Fusion Oncogenes in Pakistani Children with Acute Lymphoblastic Leukemia and Their Association with Clinical Pattern and Treatment Outcome

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5124-5124
Author(s):  
Zafar Iqbal ◽  
Sabir Noreen ◽  
Aleem Aamer ◽  
Awan Tashfeen ◽  
Tahir Naeem ◽  
...  
Keyword(s):  
Treatment Outcome ◽  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
White Cell Count ◽  
Total Leukocyte Count ◽  
Age Group ◽  
Response To Chemotherapy ◽  
Pediatric All

Abstract Abstract 5124 Background & Objectives: Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FGs) (Xu et al., 2012). The frequency of various FO can vary in different ethnic groups & these FGs have important implications for prognosis & treatment outcome (van-Dongen et al, 1999). Methods: We studied FGs in 101 pediatric ALL patients using Interphase FISH & RT-PCR (van-Dongen et al, 1999), & their association with clinical features & treatment outcome. Results: Five most common FGs i. e. BCR-ABL [t(9;22)], TCF3-PBX1 [t(1;19)], ETV6-RUNX1 [t(12;21)], MLL-AF4 [t(4;11)] & SIL-TAL1 (del 1p32) were found in 89/101 (88. 1%) patients. Frequency of BCR-ABL was 44. 5% (45/101) (Table 1). BCR-ABL positive patients had a significantly lower survival (43. 73 ± 4. 24 weeks) (Figure 1)& higher white cell count as compared to others except patients with MLL-AF4. The highest relapse-free survival (RFS) was documented in ETV6-RUNX1 (14. 167 months) followed closely by those cases in which no gene was detected (13/100). RFS in BCR-ABL, MLL-ASF4, TCF-PBX4 & SIL-TAL1 was less than 10 months (7. 994, 3. 559, 5. 500 & 8. 080 months respectively) (Figure 2 & 3). BCR-ABL: Frequency of occurrence was directly proportional to age (3 less than 2 year age group, 16 in the 2–7 year age group & 26 in the older than 7 group. Total leukocyte count (TLC) was higher when compared to patients with other oncogenes. Organomegaly was not common. BCR-ABL positivity was associated with low remission rates & shortened survival. ETV6-RUNX1: The median age of the patients was 1. 85 years. The gene frequency was highest in patients younger than 2 years. TLC was not very high & patients had a good prognosis. MLL-AF4: 17 patients had MLL-AF4 gene rearrangement with a median age of 9 years. Five patients were younger than 2 years, two between 2 & 7 years, & ten patients were in the 7–15 age group. Majority of our patients were older unlike the usual occurrence where most of the patients are infants. TCF3-PBX1: This FG occurs in around 2% of patients. Only two female patients were diagnosed with this translocation. Both the patients were over 2 years of age. It was associated with an inferior outcome in the context of response to chemotherapy & a higher risk of CNS relapse although small numbers preclude any firm conclusions. SIL-TAL1: Patients were older than 2 years, with the majority falling in the age range 7 to 15 years. The immunophenotype data were available in all SIL-TAL1 patients showing this fusion gene was associated with T-ALL with organomegaly being observed frequently. Discussion & Conclusion: This is the first study from Pakistan correlating molecular markers with disease biology & treatment outcome in pediatric ALL. Our study revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, in consistent with various other reports from Pakistan & rest of the world ((Iqbal & Akhtar, 2007; Faiz et al., 2011; (Gaynon et al., 1997; Iacobucci et al., 2012).) which, consequently, was associated with poor overall survival. The data indicates an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population & the development of facilities for stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Epigenetic Status Of CEBPA Promoter and Expression Of CEBPα Correlates With Molecular Genetic Subtype and CD2 Aberrant Expression In B Cell Precursor ALL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2573-2573
Author(s):  
Lucie Slamova ◽  
Julia Starkova ◽  
Karel Fiser ◽  
Eva Fronkova ◽  
Leona Rezkova Reznickova ◽  
...  
Keyword(s):  
B Cell ◽  
Early Phase ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Fisher Exact Test ◽  
Cell Precursor ◽  
Aberrant Expression ◽  
Mll Gene

Abstract CCAAT/enhancer binding protein alpha (CEBPα) is one of the crucial transcription factors involved in hematopoietic differentiation and leukemogenesis. CEBPα promotes myeloid differentiation by up-regulation of lineage specific genes and by cell proliferation arrest. Epigenetic regulation of CEBPα expression through DNA methylation has been demonstrated in acute myeloid leukemia (AML) (Figueroa et al, Cancer Cell, 2010). However, only limited data are available regarding CEBPA promoter methylation and its expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Methylation status of CEBPA promoter (-295 to -593bp upstream of the transcription start site (TSS), 24 CpG dinucleotides) was analyzed by bisulfite sequencing. Five subgroups of BCP-ALLs were analyzed: MLL gene rearranged (n=5), hyperdiploid (n=6), mBCR-ABLpos(n=5), ETV6-RUNX1pos(n=6) and other BCP-ALLs (no hyperdiploidy, MLL gene rearrangement, BCR-ABL or ETV6-RUNX1 fusion gene (“BCP-others”, n=29)). CEBPA promoter was hypermethylated in MLL-rearranged, hyperdiploid and ETV6-RUNX1pos BCP-ALL (5/5, 6/6 and 4/6 respectively). Surprisingly CEBPA promoter was hypomethylated in all mBCR-ABLpos cases (5/5). In subgroup of other BCP-ALLs both hypermethylation (10/29) and hypomethylation of CEBPA promoter (19/29) were detected (Figure 1A). In previous study we found association of CD2 (LFA-2) aberrant expression and switch to the monocytic lineage during the early phase of treatment in BCP-ALLs (Slamova et al, ASH 2012). We were interested if a possible link between hypomethylation of CEBPA promoter correlates with aberrant expression of CD2. There was a significant association between aberrant expression of CD2 antigen and hypomethylation in CEBPA promoter in BCP-others (Fisher exact test, p<0.0001). Interestingly, in the only hypomethylated ETV6-RUNX1pos case we found aberrant CD2 expression on blasts, which is exceptional in ETV6-RUNX1pos ALL. We next asked whether methylation of CEBPA promoter correlates with CEBPα expression. It is generally accepted that promoter hypomethylation is often associated with increased expression of the relevant gene. Our data prove that in general, this holds true also for BCP-ALL. However, in two genetically defined subsets we observed either high expression despite hypermethylation (MLL-rearranged ALL) or low expression despite hypomethylation (mBCR-ABLpos ALL) (Figure 1B). In BCP-others hypomethylation of CEBPA promoter was significantly associated with upregulation of myeloid antigens (CD14 and/or CD33) and downregulation of B cell marker CD19 on blasts during the first weeks of the treatment (Fisher test, p=0.0009). In summary Methylation status of CEBPA promoter correlates with genetic subtypes of BCP-ALL. The notion that hypomethylation leads to overexpression was confirmed in majority of BCP-ALLs, while in mBCR-ABLpos and MLL gene rearranged BCP-ALL it did not follow this pattern. Hypomethylation of CEBPA promoter in BCP- others correlates with CD2 expression on blasts and increased CEBPα gene expression. During the early phase of the treatment in other BCP-ALLs with hypomethylated CEBPA promoter increase of myeloid and decrease of B lymphoid markers on blasts was observed. Supported by: GACR P301/10/1877, GACR P304/12/2214, GAUK 914613, UNCE 204012, NT13462, NT12397- 4, project for conceptual development of research organization (Ministry of Health, CZ) 00064203, the FACS Aria instrument was supported by EU-Prague project CZ.2.16/3.1.00/24022 Disclosures: No relevant conflicts of interest to declare.

A NUP98-HOXD13 Leukemic Fusion Gene Leads To Aberrant Actin Localization In Dysplastic Megakaryocytes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2486-2486
Author(s):  
David L. Caudell ◽  
Benjamin Okyere ◽  
Jacob Cawley ◽  
Abdul Gafoor A. Puthiyaveetil ◽  
Bettina Heid
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Hematopoietic Malignancy ◽  
Glass Slides ◽  
Transmission Electron ◽  
Platelet Release ◽  
Actin Localization

Abstract Myelodysplastic syndrome (MDS) is a hematopoietic malignancy characterized by peripheral cytopenias due to bone marrow (BM) failure. Megakarypoiesis, megakaryocyte (MK) production, and platelet release are impaired in in some cases of MDS. Patients often have fewer, but larger circulating platelets, which have abnormal demarcation membrane systems (DMS); the DMS, which determines the number and size of platelets released, is dependent on actin formation. However, the precise role of actin during megakaryopoiesis is poorly understood. Transgenic mice that express the fusion gene NUP98-HOXD13 (NHD13) is a model for MDS and have dysplastic MKs in BM, and macro platelets in circulation. We hypothesized that expression of NHD13 disrupts actin localization during megakaryopoiesis resulting in reduced platelet release and macro platelet formation. To test the hypothesis, BM from wild type (WT) and NHD13 mice were flushed and cultured in media supplemented with Thrombopoietin for 5 days. Following in vitro propagation, MKs were harvested over a discontinuous gradient for downstream experiments. Sternums were also fixed in paraformaldehyde, stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 BM contained many dysplastic MKs. Harvested MKs and BM cores from one femur were processed and analyzed by transmission electron microscopy (TEM) and the ultrastructural properties of the DMS detailed. TEM of MKs showed NHD13 leads to formation of an irregular DMS along with abnormal distribution of unusually large granules in MK cytoplasm. Cultured MKs were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin and Myosin IIa and the cytoskeleton visualized by confocal microscopy. WT MKs in vitro had two phenotypes: (1) MKs with myosin and actin evenly dispersed in the cytoplasm and (2) MK with actin predominantly in the periphery of the cytoplasm. In contrast, transgenic MKs displayed only the former phenotype suggesting that actin localization is impaired in NHD13 MKs. Finally, MKs were stimulated with estrogen and adhered to fibrinogen matrices to determine their proplatelet formation functionality. Our results showed impaired proplatelets formation in NHD13 MKs. These data suggest that expression of NHD13 leads to aberrant actin localization leading to dysplastic MK differentiation and macro platelet release. Understanding molecular mechanisms of abnormal megakaryopoiesis in MDS is important as many MDS patients die of hemorrhagic complications. Further studies using this model system will provide a platform for translational research and should reveal potential therapeutic targets in MDS, leading to improved patient care/survival. Disclosures: No relevant conflicts of interest to declare.

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