Aurora-A Kinase Expression In Untreated Patients With Chronic Lymphocytic Leukemia

Abstract Aurora-A kinase is a cell-cyle regulating kinase required for chromosomal segregation. Overexpression of Aurora-A kinase has been detected some solid tumors and hematological malignancies such as multiple myelomai non-Hodgkin's lymhoma, and acute leukemia. But there are only two studies in chronic lymphocyic leukemia (CLL). This prospective study was approved by Ethical Committee of University. We investigated Aurora-A kinase in bone marrow of 41 untreated patients (22 male and 19 female with mean age of 70±10 years) with CLL and 19 patients (8 male and 11 female with mean age of 54±20 years) with anemia such as megaloblastic, autoimmune hemolytic, and iron deficiency anemia using a quantitative reverse transcriptase-PCR (RT-PCR) method. β-Actin and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA was used as the internal controls. Total RNA was extracted from bone marrow cells using the Trizol method (High Pure Isolation Kit, Roche Diagnostics). cDNA was prepared with the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). Aurora-A cDNA was quantified using TaqMan Universal PCR Mastermix (Applied Biosystems) and the Aurora-A TaqMan Gene Expression Assay. β-Actin was assayed using TaqMan Universal PCR Mastermix with forward (CCCTGGCACCCAGCAC) and reverse (GCCGATCCACACGGAGTAC) primers at 400nM each and probe (fam-ATCAAGATCATTGCTCCTCCTGAGCGC-bhq) at 100nM concentrations. Real-time quantitative RT-PCR was performed in LightCycler 480II (Roche Diagnostics). Relative RNA level was reported via standard delta delta Ct (dd Ct). Immunhistochemical analysis was performed using formalin-fixed, parafin-embedded sections of bone marrow biopsy specimens from patients and controls. Tissue sections were incubated for 60 minutes with Aurora-A (Novus Biologicals Inc. Littleton CO, 1:100 dilution). Aurora-A kinase is establish as positive if exist >10% in the cytoplasms and nuclei of the neoplastic cells in all cases. If this staining is 0-10% of cells, it is accepted as slight positive. If these cells is never (0%) stained, it is negative. For the comparison of values, Mann-Whitney-U, Chi-square, and One-Way ANOVA tests were used by SPSS 15.0 for Windows. With FISH method, 17p and 13q deletions were detected in 10% and 37% of the patients CLL, respectively. There was trisomy 12 in 7% of teh patients. 68% of the patients with CLL were in Binet-A stage. By immunohistochemical analysis, while Aurora-A kinase was positive in 61% of the patients, it was negative in 72% and slight positive 28% of controls. These positivity was statistically significant (p<0.001). By RT-PCR, β-Actin and GAPDH mRNA values were 3.85±2.61 and 3.49±2.32 in the patients with CLL, respectively. These values were 3.80±2.73 and 4.34±2.36 in controls, respectively. There was no difference for both β-Actin and GAPDH mRNA values between two groups (p>0.05). According to Binet classification, there was no difference for Aurora-A kinase expression with RT-PCR and immunohistochemical staining (p>0.05). Moreover there was no difference for both expression of Aurora-A kinase and immunhistochemical staining in between the patients with chromosmal ambnormalities and without (p>0.05). Although overexpression of Aurora-A kinase expression was not detected, significant immunohistochemical staining represented that Aurora-A kinase can be potential therapeutic target in CLL. Disclosures: No relevant conflicts of interest to declare.

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Aurora A Kinase Is Required for Hematopoiesis

Blood ◽

10.1182/blood.v120.21.1201.1201 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1201-1201

Author(s):

Benjamin Goldenson ◽

Jeremy Q Wen ◽

John Crispino

Keyword(s):

Bone Marrow ◽

Steady State ◽

Small Molecule ◽

Dna Content ◽

Colony Formation ◽

Conflicts Of Interest ◽

Aurora A Kinase ◽

Aurora A ◽

Wild Type ◽

Significant Difference

Abstract Abstract 1201 In acute megakaryocytic leukemia (AMKL), there is a failure of megakaryocytes to differentiate, become polyploid and stop dividing. We used an integrated screening approach that included chemical, proteomic and genetic screens to identify small molecules and their targets that control polyploidization and differentiation of normal and malignant megakaryocytes. We identified several small molecule inducers of polyploidy and used siRNA and proteomic target ID approaches to determine the cellular targets of the lead small molecule dimethylfasudil (diMF). Aurora Kinase A (AURKA) was identified as one of the top targets of diMF. AURKA is an attractive target in AMKL for several reasons. First, AURKA is overexpressed in AMKL cells. Second, at the biologically effective doses used in our cell-based assays, AURKA inhibition was selective for the megakaryocyte lineage. Third, AURKA inhibition by diMF or the selective AURKA inhibitor MLN8237 increased MK polyploidy, induced features of differentiation, blocked proliferation of AMKL blasts, and improved survival in an AMKL mouse model. AURKA is important in mitotic spindle assembly, mitosis, chromosomal alignment and segregation. Moreover, it is required for embryonic development, as Aurka−/− embryos fail to grow beyond the blastocyst stage. However, the extent to which AURKA is necessary for steady state hematopoiesis in adults is unknown. To investigate the necessity of AURKA in hematopoiesis, we utilized a conditionally targeted strain of mice (Aurkaflox/flox). To delete AURKA in megakaryocytes ex vivo, Aurkaflox/flox bone marrow cells were expanded, transduced with a retrovirus expressing Cre and GFP, and then cultured in the presence of THPO for 72 hours. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased DNA content. Assays for apoptosis by Annexin V staining of Aurkaflox/flox cells infected with Cre also showed increased apoptosis in AURKA-deleted cells at 24 and 48 hours. To delete AURKA in vivo, we crossed Aurkaflox/flox mice to MX1-Cre mice and injected wild-type, heterozygous and homozygous floxed mice expressing MX1-Cre with pIpC every other day for six days. We found that deletion of AURKA in hematopoietic progenitors leads to pancytopenia, profound bone marrow defects and death within two weeks. Colony formation assays showed significantly decreased myeloid, erythroid and megakaryocyte colony formation with AURKA deficiency. Bone marrow histology displayed markedly hypocellular marrow, but curiously, flow cytometry revealed a significant increase in the percentage of CD41 and CD42 positive cells. This observation suggests that AURKA normally acts to restrain terminal differentiation of megakaryocytes and is consistent with the CD41 and CD42 inducing ability of AURKA inhibitors. To confirm that AURKA is the key target of our recently identified polyploidy inducers, we assayed the effects of diMF and MLN8237 on Aurka+/+, Aurka+/− and Aurka−/− megakaryocytes. 300 nM diMF and 100 nM MLN8237, concentrations that strongly induce polyploidy, did not increase MK polyploidization in Aurka−/− MKs. diMF and MLN8237 treatment increased polyploidy in Aurka+/− MKs with no significant difference in comparison to Aurka+/+ MKs. We also assayed the ability of wild-type or the T217D mutant of AURKA, which is resistant to inhibition by MLN8237, to reduce the induction of polyploidy caused by diMF and MLN8237 upon overexpression. CMK cells were infected with viruses harboring wild-type or T217D AURKA, treated with DMSO, 3 μM diMF or 30 nM MLN8237 for 72 hours, and then evaluated for DNA content. The increase in polyploidization induced by both compounds was significantly decreased in cells overexpressing the T217D mutant of AURKA. With overexpression of the wild-type AURKA, there was a trend towards reduction in polyploidy, but more variable effects and no significant difference. Thus, AURKA T217D overexpression reduced the ability of diMF and MLN8237 to induce polyploidization, consistent with our conclusion that diMF targets AURKA. Together, our data support a role of AURKA in megakaryocyte polyploidization and differentiation and show that AURKA is required for steady state hematopoiesis. The results also show that AURKA is the key target of diMF in the induction of polyploidization of megakaryocytes and support the development of Aurora A kinase inhibitors in clinical trials for AMKL. Disclosures: No relevant conflicts of interest to declare.

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Three Novel AML Cases Harboring the Semi-Cryptic t(7;21)(p22;q22) Translocation Expressing RUNX1-USP42 Fusion Transcripts Associated with Diploidy/Tetraploidy and/or 5q Alterations: a Probably Underestimated Combination

Blood ◽

10.1182/blood.v116.21.2708.2708 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2708-2708

Author(s):

Eric Jeandidier ◽

Carine Gervais ◽

Isabelle Radford-Weiss ◽

Catherine Gangneux ◽

Valerie Rimelen ◽

...

Keyword(s):

Bone Marrow ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Fusion Transcript ◽

Fish Analysis ◽

Chimeric Transcript ◽

Rt Pcr ◽

Balanced Translocation ◽

Fusion Transcripts ◽

Cryptic Translocation

Abstract Abstract 2708 RUNX1 is implicated in numerous chromosomal abnormalities acquired in acute myeloid leukemia (AML). The most frequent one, the t(8;21) is associated with a particular morphology together with a favorable prognosis. This is not the case for other 21q abnormalities, that are much less frequent and for which the prognosis is quite different. Moreover, beside point mutations, conventional cytogenetics failed to detect some of chromosomal alterations involving RUNX1. Recently 3 cases of the rare and semi-cryptic t(7;21)(p22;q22) translocation expressing the RUNX1-USP42 fusion transcripts have been reported, demonstrating the recurrence of this abnormality in AML. We describe here 3 additional cases with the same translocation and fusion transcripts, associated to 5q alterations leading to EGR1 and CSF1R heterozygous losses. In all our patients, the t(7;21)(p22.1;q22.3) was initially detected by the systematic FISH evaluation of the blastic populations using ETO-AML1 Dual Fusion probe. Patient#1 bone marrow karyotype was characterized by a tetraploid clone (89,XXYY) with loss of chromosomes 15, 17 and 18 in addition to the t(7;21), and a unbalanced translocation der(5)t(5;13)(q23;q?) between long arms of chromosomes 5 and 13, resulting in a heterozygous loss of EGR1 and CSF1R. Patient #2 blood and bone marrow karyotypes revealed a diploid clone with a del(5)(q31q33) associated with the t(7;21). The FISH analysis confirmed EGR1 and CSF1R deletions. In patient #3, the bone marrow karyotype showed diploid/tetraploïd clones, both harboring the t(7;21)(p22;q22), confirmed by FISH experiments (WCP7, AML1 probes). In addition, a der(5)t(1;5)(q3?2;q21-23) was identified within the tetraploïd clone, resulting in the loss of EGR1 and CSF1R, confirmed by FISH. In all three cases a RUNX1-USP42 fusion transcript was detected using RT-PCR, as well as the reciprocal transcript. Sequence analysis of RT-PCR products showed that the breakpoints occurred exactly in the same introns of USP42 and RUNX1 as in the previously described cases. For patient #1 and #3 a chimeric transcript was found formed of the RUNX1 exon 7 fused to the USP42 exon 3. In patient #2, a shorter chimeric transcript arised from the fusion of the RUNX1 exon 5 to the exon 3 of USP42. As already noticed in the previous reports, an alternative splicing of the RUNX1 exon 6 has been detected in these three cases. The description of these 3 novel t(7;21) confirm the recurrence of this balanced translocation in AML, and shows that this chromosomal abnormality is often associated with diploid/tetraploid clones and/or 5q alterations. Special attention should be paid in karyotype analysis of AML with diploid or tetraploid clones harboring 5q alterations. In such cases RUNX1 rearrangements should be explored using FISH analysis, and RUNX1-USP42 fusion transcript should be searched by RT-PCR in positive cases. Prospective and retrospective studies of AML have now to be settled in order to assess the incidence and clinical relevance of this cryptic translocation. Disclosures: No relevant conflicts of interest to declare.

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Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia

Blood ◽

10.1182/blood.v116.21.4435.4435 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4435-4435

Author(s):

Rong Fu ◽

Hui Liu ◽

Zonghong Shao ◽

Jun Wang ◽

Lijuan Li

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Mononuclear Cell ◽

Rt Pcr ◽

Glycine Buffer ◽

Healthy Donors ◽

Preliminary Study ◽

The Relationship ◽

Epor Expression ◽

Normal Controls

Abstract Abstract 4435 Objective To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's (BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP), and then explore the probable autoantigens of auto-Ab in IRP. Methods 46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy donors as controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with flow cytometry to observe the relationship between EPOR and auto-Ab; After sorting erythropoietic cells in bone marrow, EPOR mRNA and protein Stat5,P-Stat5 were investigated by RT-PCR and Western blot to observe the production of EPOR and EPO/EPOR signal transduction; Finally, EPOR expression on the membrane were tested again after stripping auto-Ab with glycine buffer. Results (1) EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (-) arm(4.58±4.09)%(P<0.01), and the latter was significantly higher than that of normal controls (2.27±1.76)%(P<0.05); EPOR of IRP patients was negatively correlated with their auto-Ab (r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(auto-Ab);(2)EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significantly higher than that of auto-Ab (-) arm(0.554±0.116)(P<0.01)and normal controls (0.580±0.119)(P<0.05);(3)Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls (0.54±0.36)(P<0.05); While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls (0.85±0.38)(P<0.05); (4) EPOR expression became higher while auto-Ab became lower after stripping with glycine buffer. Conclusion The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR was one of autoantigens in IRP. Disclosures: No relevant conflicts of interest to declare.

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Expression of dlk1 in the Bone Marrow Cells of Patients with Myelodysplastic Syndrome and Its Clinical Significance

Blood ◽

10.1182/blood.v118.21.5042.5042 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5042-5042

Author(s):

Zonghong Shao ◽

Lanzhu Yue ◽

Rong Fu ◽

Lijuan Li ◽

Erbao Ruan ◽

...

Keyword(s):

Bone Marrow ◽

Early Diagnosis ◽

Myelodysplastic Syndrome ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Rt Pcr ◽

Normal Controls ◽

Lower Expression ◽

Bone Marrow Blasts ◽

Marrow Cells

Abstract Abstract 5042 Objective To investigate the expression of dlk1 gene (delta-like 1) in the bone marrow cells of patients with Myelodysplastic syndrome (MDS), and explore the molecular marker for early diagnosis of MDS. Methods The expression of dlk1 mRNA in the bone marrow cells of cases with MDS, AML and normal controls were measured by RT-PCR, aiming to search for the cytogenetic marker of MDS malignant clone. Results The expression of dlk1 mRNA in bone marrow cells of MDS patients (0.7342±0.3652) was significantly higher than that of normal controls (0.4801±0.1759) (P<0.05), and was significantly positively correlated with the proportion of bone marrow blasts(r=0.467,P<0.05). The expression of dlk1 mRNA significantly increased as the subtype of MDS advanced (P<0.05). Patients with abnormal karyotypes displayed significantly higher expression of dlk1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Patients with higher expression of dlk1(≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower expression (<0.8) of dlk1 (0.1517±0.3109) (P<0.05). Conclusion dlk1 gene was highly expressed in MDS patients, which increased as the subtype of MDS advanced. The expression of dlk1 mRNA was significantly positively correlated with the proportion of bone marrow blasts. High expression of dlk1 gene suggests high malignant clone burden of MDS. Disclosures: No relevant conflicts of interest to declare.

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Phagocytosis-Mediated Acute Hemolytic Events Induce Distinct Responses By Inflammatory Monocytes

Blood ◽

10.1182/blood.v126.23.3563.3563 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3563-3563

Author(s):

Lyla A Youssef ◽

Stuart Phillip Weisberg ◽

Sheila Bandyopadhyay ◽

Eldad A. Hod ◽

Steven L Spitalnik

Keyword(s):

Bone Marrow ◽

Inflammatory Response ◽

Conflicts Of Interest ◽

The United States ◽

Dependent Manner ◽

Rt Pcr ◽

Monocyte Chemoattractant Protein 1 ◽

Inflammatory Monocytes ◽

Reporter Mice ◽

Red Pulp

Abstract Introduction Red blood cell (RBC) transfusions are a common therapy, with ~15 million RBC units administered annually in the United States. Studies in mice and dogs identified increased circulating levels of multiple inflammatory cytokines after transfusions of refrigerator storage-damaged RBCs. One chemokine identified was monocyte chemoattractant protein 1 (MCP-1; also known as CCL2). MCP-1 is the ligand for CCR2, which is expressed by several cell types including Ly6Chi (i.e., inflammatory) monocytes. One reserve site of Ly6Chi monocytes is the bone marrow, from which they emigrate into the circulation in a CCR2-dependent manner to traffic to sites of inflammation. Because the spleen is a primary site of RBC clearance, we aimed to identify splenic cells responsible for producing MCP-1 following transfusions of storage-damaged or antibody-coated RBCs. Methods Leukoreduced murine RBCs were prepared from wild-type (WT) C57BL/6 donors and refrigerator stored for 12-13 days. MCP-1 transgenic reporter mice were transfused with fresh, or storage-damaged ("old"), or fresh anti-RBC antibody-coated RBCs. Spleen, bone marrow, and blood were collected post-transfusion and cells were analyzed by multi-parameter flow cytometry. WT C57BL/6 mice were also transfused with fresh or old RBCs; splenocytes isolated post-transfusion were flow sorted followed by RNA isolation and RT-PCR. Results Splenic red pulp macrophages (VCAM1hi, F4/80hi, CD11blo) were primarily responsible for erythrophagocytosis after transfusions with old or antibody-coated RBCs. However, in each case, Ly6C hi, CD11b+ splenic inflammatory monocytes in MCP-1 reporter mice expressed MCP-1. MCP-1 expression by these cells was also confirmed in WT recipients by RT-PCR after flow sorting. Interestingly, only a small percentage of inflammatory monocytes ingested transfused RBCs. Furthermore, circulating inflammatory monocyte levels increased following transfusion of old or antibody-coated RBCs, accompanied by reduced levels in the bone marrow. Conclusions Although red pulp macrophages were the major cell type responsible for clearing transfused refrigerator-damaged or antibody-coated RBCs, a different splenic cell population (i.e., inflammatory monocytes) produced MCP-1. Thus, the splenic reserve of inflammatory monocytes produced an inflammatory response following phagocytosis-mediated acute hemolytic events. Increased numbers of circulating inflammatory monocytes and reduced numbers in the bone marrow suggest that these cells emigrated from the bone marrow, perhaps in response to MCP-1 signaling. Thus, one possible mechanism explaining our results is that erythrophagocytosis in the spleen induces an inflammatory response, triggering splenic inflammatory monocytes to synthesize MCP-1 and release it into the circulation; MCP-1 then binds CCR2 on bone marrow inflammatory monocytes, causing their egress into the circulation. The additional signals involved in these phenomena, along with their clinical relevance to transfusion medicine, require additional investigation. Disclosures No relevant conflicts of interest to declare.

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Thrombocytopenia In Severe Anemia of Iron Deficiency

Blood ◽

10.1182/blood.v116.21.5153.5153 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5153-5153

Author(s):

Getinet D. Ayalew ◽

Juhi Mittal ◽

Ratesh Khillan ◽

Miriam Kim ◽

Albert S. Braverman ◽

...

Keyword(s):

Bone Marrow ◽

Iron Deficiency ◽

African American Women ◽

Conflicts Of Interest ◽

Ferrous Sulphate ◽

Bone Marrow Aspiration ◽

Deficiency Anemia ◽

Platelet Counts ◽

Cell Counts ◽

Erythroid Hyperplasia

Abstract Abstract 5153 Introduction: Iron deficiency suppresses hemoglobin synthesis and erythropoiesis, but the resulting anemia is frequently associated with thrombocytosis. Methods: The clinical and hematologic data of seven women with severe iron deficiency anemia (IDA) and thrombocytopenia were retrospectively analyzed. Results: All patients were African-American women with symptomatic IDA, due to bleeding from uterine fibroids in 6 and from colonic diverticulosis in 1. They were 31–70 years of age, median 38. None had palpable splenomegaly. Hemoglobin ranged from 2.9–5.5, median 4.2 g/dL. MCV ranged from 57–70 fl, median 68. Absolute reticulocyte counts ranged from 19,000 – 23, 000/mm3. The initial serum ferritin ranged from 2 to 42 ng/ml, median 4. Serum iron levels ranged from 10 to 70 mcg/dl with median 30, while iron-binding capacities ranged from 381–426 mcg/dl. Serum erythropoietin (EPO) levels were >2000U/ml in two of the patients. Serum lactic dehydrogenase, bilirubin levels and liver function tests were normal; and Coombs' test negative in all cases. White blood cell counts were normal. The platelet counts ranged from 12 to 103, with a median of 46 × 109/L. Peripheral blood smears showed microcytic hypochromic red blood cells (RBC), with no evidence of platelet clumping. Bone marrow aspiration and biopsy on two patients showed increased numbers of normal megakaryocytes, erythroid hyperplasia and absent iron stores. Six patients were treated with packed RBC transfusions, and ferrous sulphate 325 mg orally was initiated at presentation in 7. Their thrombocytopenia was not treated with steroids or other agents. Three patients' platelet count reached normal or super-normal levels within 72 hours. Six patients were seen at ≥3 months after presentation, and all had achieved normal platelet counts and hemoglobin. Conclusions: These data imply that severe IDA can sometimes cause thrombocytopenia rather than thromobocytosis. We cannot be sure whether these patients' uniform normalization of platelet counts was due to treatment of their anemia by transfusion, or iron therapy. Though bone marrow megakaryocyte numbers were increased in 2 patients, there is no evidence for peripheral platelet destruction. Platelet release from megakaryocytes may have decreased in these patients. Pharmacologic EPO therapy can occasionally cause thrombocytopenia, and high endogenous EPO levels in our patients may have reduce their platelet counts. This conclusion is consistent with their apparent response to transfusion. Though the pathogenesis of IDA-associated thrombocytopenia is not known, our data suggest that the results of anemia and iron deficiency treatment should be evaluated before investigating thrombocytopenia as an independent problem. Disclosures: No relevant conflicts of interest to declare.

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Molecular Characterization and Clinical Correlations of MEK1/2 Inhibition (AZD6244) in Relapse or Refractory Multiple Myeloma: Analysis From a Phase II Study

Blood ◽

10.1182/blood.v118.21.306.306 ◽

2011 ◽

Vol 118 (21) ◽

pp. 306-306 ◽

Cited By ~ 1

Author(s):

Adriana Zingone ◽

Neha Korde ◽

Jinqiu Chen ◽

Liqiang Xi ◽

Mark Raffeld ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Phase Ii ◽

Partial Remission ◽

Drug Exposure ◽

Conflicts Of Interest ◽

Molecular Evidence ◽

Rt Pcr ◽

Mek Inhibition ◽

Qrt Pcr

Abstract Abstract 306 Gene expression profiling previously defined 7 molecularly distinct multiple myeloma (MM) subgroups associated with clinical behavior. The MMSET/FGFR3 subgroup, harboring the t(4;14) translocation, and the MAF subgroup including t(14;16) (c-MAF), t(14,20) (MAF-B) and 8q24.3 (MAF-A), were associated with the shortest progression-free and overall survival. Recently, we demonstrated that MM cell lines of the MMSET/FGFR3 (MS) and MAF (MF) subgroups are dependent on MEK signaling for growth and survival. Inhibition of mitogen-activated protein (MAP) kinase pathway, by genetic or pharmacologic means, specifically decreased expression of MAF and its target genes. In addition, MEK inhibition enhances myeloma cell apoptosis in the presence of standard therapeutic agents due to the effects on the bone marrow microenvironment, suggesting that MEK inhibitors could kill cells resistant to conventional therapies. As part of our ongoing phase II multi-center clinical trial testing the MEK inhibitor AZD6244 in patients with relapsed or refractory MM, we have conducted extensive molecular profiling for a subset of patients, to characterize the underpinnings of MEK inhibition and its correlation with clinical outcomes. To date, of the 37 patients enrolled on trial, 12 patients consented to correlative sampling, including bone marrow aspirates performed at baseline and day 2 after initiation of AZD6244 treatment (75 mg twice daily, continuously on a 28 day cycle). CD138+ cells were purified using magnetic beads (Miltenyi) and subjected to RNA, DNA and protein extraction following standard procedures. qRT-PCR and RT-PCR was performed to detect the expression level of MAF genes (c-MAF, MAF-B, MAF-A ) and the presence of the JH-MMSET hybrid respectively. Nanopro-immunoassay was used to assess the level of total BIM, total and phospho-MEK1/2 and ERK1/2 pre- and post-treatment. Pyrosequencing was performed to detect the presence of mutations in RAS and BRAF genes. At this time, 11 patients have been molecularly characterized and included in this analysis. We found 3 (27%), 2 (18%) and 1 (9%) patients with a KRAS, NRAS, and BRAF mutation, respectively. Based on 8 patients analyzed to date, qRT-PCR showed that 4 (50%) over-expressed c-MAF or MAF-B genes; results from the MAF-A analysis are currently pending. RT-PCR detected the presence of JH-MMSET hybrid in one patient. At this time, we have detailed clinical data for 8 patients: one patient (MS) had a very good partial remission and the duration of response (DOR) was 8 months; one patient (MAF-B) had a partial remission with a DOR of 6 months; and two patients (one MAF-B, one with pending results) had stable disease with DORs of 5+ and 13 months. In these patients, high levels of phospho-ERK1/2 were detected at baseline, and were dramatically reduced after AZD6244 exposure. Also, MEK inhibition resulted in increased levels of total ERK1/2 and MEK1/2 as well as pro-apoptotic BIM. In subanalysis, CD138+ cells obtained from an extramedullar plasmacytoma in the patient carrying the MMSET translocation were treated in vitro with AZD6244; this resulted in undetectable levels of phospho-ERK1/2 post drug exposure. Among this group of relapsed MM patients, refractory to several lines of therapy and with molecular evidence for myeloma cells being MEK dependent, we found single drug AZD6244 to result in durable responses (up to 13 months). Our novel results provide molecular and clinical insight on MEK inhibition in MEK-dependent myeloma tumor cells, and support development of rational drug allocation in MM. Disclosures: No relevant conflicts of interest to declare.

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Whole Exome Sequencing Identifies a Novel Variant in the Pklr Gene Which Worsen the Anemia Status in a Congenital Dyserthropoietic Anemia Patient

Blood ◽

10.1182/blood-2018-99-112743 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4880-4880

Author(s):

Ching-Tien Peng

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Conflicts Of Interest ◽

Deficiency Anemia ◽

Sequence Variant ◽

Variable Degree ◽

Novel Variant ◽

Sanger Sequence ◽

Hereditary Disorders ◽

Chronic Hemolysis

Abstract Congenital dyserythropoietic anemia (CDAs) is a group of hereditary disorders characterized by ineffective erythropoiesis and distinct morphological abnormalities of erythroblasts in the bone marrow. Diagnosis of CDA is based primarily on the morphology of bone marrow erythroblasts but genetic tests might have more and more important roles recently. Here, we describe a 31-year old female with atypical CDA under regular blood transfusions. The results of blood tests were as followed: RBC: 3080000/µL, Hb: 9.9 g/dL, Hct: 26.8%, MCV: 87.0 fl, MCH:32.2 pg, and MCHC: 36.9 g/L. In this study, analysis of CDA-related genes, including SEC23B, CDAN1, KLF1, and C15orf41, were analyzed by exome sequencing but no pathogenic variant was found. In additional, we analyzed RBC-related genes and a novel variant in the PKLR p.A468G (NP_000289) was found which was also confirmed by Sanger sequence. Variant of PKLR gene has been reported as the cause of pyruvate kinase (PK) deficiency anemia. PK deficiency is the most cause of congenital nonspherocytic hemolytic anemia with GMAF (global minor allele frequency) of 0.0001. The clinical features of PK-deficient patients are highly variable degree of chronic hemolysis with severe neonatal jaundice and fetal anemia at birth. In this case, PKLR p.A468G heterozygous variant was detected in a patient with PK deficiency, as an example of precision diagnosis by exome sequencing. Disclosures No relevant conflicts of interest to declare.

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Research of Gene Expression in Patients with Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v114.22.4433.4433 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4433-4433

Author(s):

Bao-An Chen ◽

Bo Zhang ◽

Chong Gao ◽

Guo-Hua Xia ◽

Ze-ye Shao ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Clinical Significance ◽

Myelodysplastic Syndromes ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Bone Marrow Mononuclear Cells ◽

Rt Pcr ◽

Normal People ◽

Significant Difference

Abstract Abstract 4433 Object This study was aimed to investigate the expression of c-FLIPL, c-FLIPS and DLK1 mRNA in Myelodysplastic Syndromes (MDS) patients, as compared with normal people and AML patients, and to find its clinical significance. Methods The expression of c-FLIPL, c-FLIPS and DLK1 mRNA in bone marrow mononuclear cells (BMNNC) of 16 patients with MDS, 8 patients with AML and 3 controls were detected by RT-PCR. Results The expression of DLK1 mRNA was up-regulated in MDS, including RA and RAEB, as compared with controls(P<0.05). There was no significant difference in expression of DLK1 between RA and RAEB(P>0.05). The expression of DLK1 was significant higher in AML patients, compared with controls(P<0.05). There was no significant difference between MDS and AML patients(P>0.05). The expression of c-FLIPL mRNA was higher than that in controls, both in RA and RAEB(P<0.05). There was no significant difference in expression of c-FLIPL between RA and RAEB(P>0.05). In eight AML patients, c-FLIPL gene's expression was up-regulated, as compared with controls(P<0.05). Between AML and MDS patients, there was no significant difference(P>0.05); The expression of c-FLIPS mRNA had no significant difference between MDS patients and controls(P>0.05), but its expression in RAEB was significant higher as compared with RA patients and controls(P<0.05). And in AML patients, the expression of c-FLIPS was higher than that in controls(P<0.05), but there was no significant difference between AML and MDS patients(P>0.05). Conclusion It is concluded that the expressions of DLK1, c-FLIPL and c-FLIPS mRNA in MDS/AML patients are abnormal as compared with normal people, although there are no significant difference have been found between AML and MDS. These genes may play critical roles in escaping malignant clone of MDS from apoptosis and acquiring the ability to divide unlimitedly, they can become important indexes for evaluating of development in MDS. Disclosures: No relevant conflicts of interest to declare.

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TET2 mRNA Expression in Bone Marrow Mononuclear Cells of the Patients with Myelodysplastic Syndromes and it's Clinical Significances

Blood ◽

10.1182/blood.v118.21.5036.5036 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5036-5036

Author(s):

Zonghong Shao ◽

Wei Wang ◽

Rong Fu ◽

Jun Wang ◽

Lijuan Li ◽

...

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Lower Proportion ◽

Bone Marrow Mononuclear Cells ◽

Rt Pcr ◽

Important Indicator ◽

Significant Difference ◽

Lower Expression

Abstract Abstract 5036 Objective This study was aimed to investigate the expression of TET2 mRNA in the bone marrow mononuclear cells(BMMNC)of patients with myelodysplastic syndrome(MDS)and its clinical significance. Methods The mRNA expression of TET2 in bone marrow mononuclear cells(BMMNC) of 25 patients with MDS and 16 controls were detected by RT-PCR. Results The expression of TET2 mRNA in BMMNC was down-regulated in MDS (0.9509±0.3841)compared with that in controls(1.2515±0.3749)(P<0.05), but was no significant difference of BMMNC expression of TET2 among RA, RCMD and RAEB. Patients with higher expression of TET2(≥0.9) presented significantly lower proportion of bone marrow blasts[(1.04±1.68)%] than that [(6.13±8.17)%] of those with lower expression (<0.9) of TET2 (P<0.05). The expression of TET2 mRNA in BMMNC of MDS patients was inversely correlated with malignant clone burden (r=-0.398,P<0.05) and IPSS (r=-0.480,P<0.05). Conclusions The mRNA expression of TET2 in BMMNC of MDS patients decreased, which might useful as an important indicator for the evaluation of MDS clone burden. Disclosures: No relevant conflicts of interest to declare.

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