ALBERT

Description: Acute erythroid leukemia (AEL) is a distinct subtype of acute myeloid leukemia (AML) characterized by predominant erythropoiesis. Currently, only few studies using next-generation sequencing were reported in AEL. To decipher the somatic mutation spectrum and discover disease-driving genes responsible for the pathogenesis of AEL, we performed whole exome-sequencing (WES) in 6 AEL and validating using targeted next generation sequencing (NGS) and Sanger sequencing in 58 AEL. From August 2003 to October 2014, a total of 158 patients fulfilling the WHO criteria for AEL were identified, comprising 91 males and 67 females. Median age was 50 years. These patients were further subclassified into 3 groups: 37 AEL after MDS, 108 de novo AEL, and 13 AML with myelodysplasia-related changes. In total, we identified 52 genes with somatic mutations in at least 2 patients, including CEBPA in 4 patients and GATA2 in 2 patients. We identified high frequencies of mutations in CEBPA (40.0%, 22/55; about 1/4 are biallelic mutations), GATA2 (22.4%, 13/58), NPM1 (15.5%, 9/58), SETBP1 (12.1%, 7/58), and U2AF1 (12.1%, 7/58), followed by TP53 (5.2%, 3/58), RUNX1 (3.5%, 2/58), TET2 (3.5%,2/58), ASXL1 (3.5%, 2/58), DNMT3A (3.5%, 2/58), SRSF2 (1.7%, 1/58) and FLT3 (1.7%, 1/58). We did not detect alterations of some of commonly mutated genes associated with AML, including IDH1, IDH2 and RAS. The results are summarized in Figure 1. To further identify the prevalence of GATA2 mutations in hematologic malignancies, we amplified and sequenced the entire coding region of GATA2 gene in 253 non-AEL AML, 40 chronic myeloid leukemia in blast crisis (CML-BC), 55 B-cell acute lymphoblastic leukemia (B-ALL), and 38 T-cell acute lymphoblastic leukemia (T-ALL). We detected GATA2 mutations in 5.5% of non-AEL AML, 15% of CML-BC, and none of B-ALL or T-ALL. The GATA2 mutations in AEL are mainly localized in ZF1 domain (P304H, D309E, A318V/T, G320S/D, L321P, and R330X) and TAD domain (Q20H). To find out the implications of GATA2 mutations in the leukemogenesis of AEL, we overexpressed the mutants of GATA2 (P304H, L321P, and R330X) in 293T cells and demonstrate that GATA2 mutant led to reduced transcriptional activity. Whereas overexpression of GATA2 mutants in mouse myeloid progenitor cell line, 32D, has no effect on the proliferative or colony formation abilities, it caused increased expression of erythroid-related antigen Ter-119 (Figure 2), b-globin and bh1-globin. Furthermore, 32D cells transfected with GATA2 mutants showed increased positivity than control cells by Benzidine staining. Taken together, our findings demonstrate that the mutatome of AEL is different from other types of AML. AEL is associated with a high frequency of mutations in GATA2 and CEBPA. GATA2 mutations resulted in a decrease of transcriptional activity and erythroid development of mouse myeloid progenitors, suggest an important role of GATA2 mutations in AEL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Myelodysplastic syndrome (MDS) is a group of hematopoietic stem/progenitor cell clonal disorders characterized by dysplastic morphology of at least one cell lineage and high risk of evolution to acute myeloid leukemia (AML). Thus, it could be viewed as a preneoplastic stage prior to development of overt acute myeloid leukemia (AML). A number of studies indicated that oncogene-induced senescence as a crucial cellular response at premalignant stage can protect cells from tumor formation. Senescent cells emerged in premalignant cell population but not in malignant ones. p16INK4a have been identified as a cellular senescence-associated molecular marker. In present study, we have determined and compared the expression level of p16INK4a by quantitative RT-PCR and western blot in BM mononuclear cells (BMMNCs)/CD34+ cells among 53 patients of MDS and 12 of AML as well as 11 healthy controls. A significantly upregulated expression level of p16INK4a in MDS, while an significantly lower p16INK4a expression level in AML had been detected in comparison with that in healthy controls(

Description: Abstract 4858 Objective To determining the clonal origin of dysplatic cells in Myelodysplastic syndromes (MDS) . Methods Karyotypic analyses of bone marrow cells using R-banding technique were carried out to determine the chromosomal abnormalities. Interphase fluorescence in situ hybridization (FISH) and morphologic analysis of bone marrow aspirates were performed in the same cells to investigate the clonal origin of dysplatic cells in 8 MDS patients. Result All patients had clonal karyotypic abnormalities: simple abnormality in 1 patient, complex abnormalities in 6 patients, coexistent of two unrelated clones in 1 patient. Most of dysplastic cells in 7 of 8 MDS patients derived from neoplasia clone while 1 patient had a reverse result,no matter what cell lineage was involved. Some of non-dysplastic cells of all patients derived from malignant clone; in 7 patients, the proportion of dysplastic cells in malignant clone were significantly higher than that of non- malignant clone. Conclusion Most of dysplastic cells in MDS derived from malignant clones, while the minority of them derived from non-malignant clones. Thus, it is reasonable to expect that in most cases myelodysplasia is present in malignant clone and can be taken as an important diagnostic evidence for MDS. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4242 A pericentric inversion(9)(p21q34) was identified in five Ph-positive leukemia patients including four patients with chronic myeloid leukemia(CML) in chronic phase(CP) and one patient with acute myeloid leukemia (AML) since 1998. Among them, two were males, three wrer females. The median age is 31 years (range 21-52 years). Conventional karyotypic analysis with R-banding technique showed that the inv(9)(p21q34) always occurred in the der(9)t(9;22)(q34;q11) and was accompanied by the der(22)t(9;22)(q34;q11) in all metaphases analyzed in four patients with CML-CP at diagnosis. One patient with AML presented three clones: one with normal karyotype, one with sole t(9;22)(q34;q11), one with inv(9)(p21q34) involving the der(9)t(9;22) and additional t(8;12)(q12;p11). FISH using LSI BCR/ABL dual-color, dual fusion probe, chromosome painting(CP) with the paint probes for chromosome 9p and 9q and RT-PCR using the primers of the BCR/ABL fusion genes were performed in four of them. FISH showed the coexistence of clone with sole t(9;22) and another with inv(9)(p11q34) and t(9;22) in four patients in whom, one patient also showed a deletion of partial sequence from BCR on der(9)t(9;22)inv(9)(p21q34). RT-PCR revealed a b3a2 transcript for BCR/ABL fusion gene transcript. FISH proved that the inv(9)(p21q34) disrupted the ABL/BCR fusion gene at the molecular level. However, it does not appear to have any biological significance because BCR/ABL fusion gene is thought to be involved in the pathogenesis of CML, while ABL/BCR fusion gene is merely mechanical consequence of the t(9;22) translocation. As far as we know, inv(9)(p21q34) has not been reperted previously. Thus, it should be regarded as a novel rare but recurrent secondary chromosomal anomaly. In this series, one lost to follow-up, one patient transformed into B cell acute lymphocytic leukemia, who and other two patients survivals of 28 days, 13 and 34 months, respectively. Only one remains alive. Their dismal outcome probably suggests inv(9)(p21q34) having unfavourable impact on prognosis. At present, no firm conclusion can be drawn from this study. More patients with this anomaly need to be investigated to elucidate its prognostic significance. Disclosures: No relevant conflicts of interest to declare.

Description: ObjectiveBased on the albumin substitution fluid, we performed plasma-free exchange in the removal of abnormal M protein for patients with multiple myeloma using Fresenius COM.TEC machine MethodsFrom February 2016 to August 2018, 70 patients with multiple myeloma, who have indication of plasma exchange because of increased M protein, were treated with plasma-free exchange. Record the relevant basic information, the clinical symptoms and related disease indicators, and use SPSS 20.0 to analyze the changes of erythrocyte sedimentation rate, blood routine, albumin, renal function, serum calcium and immunoglobulin, and report adverse reactions. ResultsAfter treatment, the patient's erythrocyte sedimentation rate, serum calcium, serum creatinine, β2-microglobulin, uric acid, IgG, IgA were significantly reduced; hemoglobin, white blood cell level were not statistically significant. The creatinine remission rate was 97%, and the platelet decline was only 17.8%, and no adverse reactions related to thrombocytopenia such as hemorrhage and ecchymoses were observed. Patients with IgG type responded better than IgA-free treatment to plasma-free exchange. There were no serious adverse reactions after treatment, 1 case of blood pressure drop, 2 cases of low calcium reaction, all in the normal range, and recovered after the end of treatment. ConclusionPlasma-free exchange is an effective technology reducing M protein, which can effectively improve the erythrocyte sedimentation index, renal function index and blood viscous state. Patients with IgG type or higher ISS stage have more obvious effects. Plasma-free exchange significantly get rid of the application of fresh frozen plasma. Disclosures No relevant conflicts of interest to declare.