Proportion Of Bone Marrow Blasts Research Articles

Gene Profile and Clinical Significance of Concomitant Mutations in CN-AML Patients with CEBPA Mutation

To analyze the occurrence of concomitant gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients with CEBPA mutation and its impact on the clinical characteristics and prognosis of the patients. 151 newly diagnosed patients with CN-AML in the Second Hospital of Shanxi Medical University from June 2013 to June 2020 were analyzed retrospectively. 34 common genetic mutations associated with hematologic malignancies were detected by next-generation sequencing technology. The occurrence of concomitant gene mutations in patients with CEBPA positive and negative groups was compared, and the correlation between concomitant mutations in different functional groups and the clinical characteristics and prognosis of CN-AML patients with CEBPA mutation was analyzed. In 151 patients with CN-AML, 55 (36.42%) were positive for CEBPA mutation (including 36 cases of CEBPAdm and 19 cases of CEBPAsm), of which 41 (74.55%) had co-mutations with other genes. The main mutated genes were GATA2 (25.45%, 14/55), TET2 (21.82%, 12/55), FLT3 (20.00%, 11/55), NRAS (12.73%, 7/55) and WT1 (9.09%, 9/55), etc. Some cases had two or more concomitant gene mutations. Grouping the mutant genes according to their functions showed that CEBPA+ group had lower mutation rates of histone methylation (P =0.002) and chromatin modification genes (P =0.002, P =0.033), and higher mutation rates of transcription factors (P =0.037) than CEBPA- group. In 55 patients with CEBPA+ CN-AML, the platelet count at diagnosis in signaling pathway gene mutation-positive group was lower than that in the mutation-negative group (P =0.005), the proportion of bone marrow blasts in transcription factor mutation-positive group was higher than that in the mutation-negative group (P =0.003), and the onset age in DNA methylation gene mutation-positive group and chromatin modifier mutation-positive group was older than that in the mutation-negative group, respectively (P =0.002, P =0.008). DFS of CEBPA+ CN-AML patients in signaling pathway gene mutation group was shorter than that in signaling pathway gene mutation-negative group (median DFS: 12 months vs not reached) (P =0.034). Compared with DNA methylation gene mutation-negative group, CEBPA+ CN-AML patients with DNA methylation gene mutation had lower CR rate (P =0.025) significantly shorter OS and DFS (median OS: 20 months vs not reached, P =0.006; median DFS: 15 months vs not reached, P =0.049). OS in patients with histone methylation gene mutation was significantly shorter than that in the histone methylation gene mutation-negative group (median OS: 12 months vs 40 months) (P =0.008). Multivariate analysis of prognostic factors showed that the proportion of bone marrow blasts (P =0.046), concomitant DNA methylation gene mutation (P =0.006) and histone methylation gene mutation (P =0.036) were independent risk factors affecting the prognosis. CN-AML patients with CEBPA mutation have specific concomitant gene profile, and the concomitant mutations of different functional genes have a certain impact on the clinical characteristics and prognosis of the patients.

Analysis of the clinical characteristics and prognosis of adult de novo acute myeloid leukemia (none APL) with PTPN11 mutations.

We discuss the clinical characteristics and prognostic significance of adult individuals with PTPN11 mutations who have developed acute myeloid leukemia (AML) (none acute promyelocytic leukemia). Next generation sequencing and Sanger sequencing were used to detect 51 gene mutations, and multiplex-PCR was used to detect 41 fusion genes from 232 de novo adult AML patients retrospectively. About 7.76% patients harbored PTPN11 mutations, 20 PTPN11 alterations were identified, all of which were missense mutations in the N-SH2 (n = 16) and PTP (n = 4) domains located in exon 3. Patients with PTPN11 mut had significantly higher platelet counts and hemoglobin levels (p < 0.001), which were mainly detected in M5 (n = 12, 66.67%, p < 0.001) subtype. Patients with MLL-AF6 positive showed a higher frequency of PTPN11 mut (p = 0.018) in the 118 AML cases. PTPN11 mut were accompanied by other mutations, which were NPM1 (44.44%), DNMT3A (38.89%), FLT3 (38.89%), and NRAS (17.2%). PTPN11 mut had a negative impact on the complete remission rate in M5 subtype patients (p < 0.001). However, no statistically significant effect on overall survival (OS) with PTPN11 mut patients in the whole cohort and age group (p > 0.05) was observed. Further analysis revealed no significant difference in OS among NPM1 mut/PTPN11 mut, NPM1 mut/PTPN11 wt, DNMT3A mut/PTPN11 mut, and DNMT3A mut/PTPN11 wt patients (p > 0.05). Multivariate analysis showed the proportion of bone marrow blasts ≥65.4% was a factor significantly affecting OS in PTPN11 mut patients (p = 0.043).

Long-term follow-up of humanized and murine CD19 CAR-T-cell therapy for B-cell acute lymphoblastic leukemia

Objective: Murine CD19 chimeric antigen receptor T-cell (CAR-T) products have been approved for the treatment of refractory/relapsed (R/R) B-cell acute lymphocytic leukemia (B-ALL) ; moreover, humanized products are also undergoing clinical trials. This study aimed to explore the differences in safety and short- and long-term follow-up efficacy between humanized and murine CD19 CAR-T-cells for treating relapsed and refractory B-ALL. Methods: Clinical data of 80 patients with R/R B-ALL treated with CD19-targeted CAR-T-cells at the Union Hospital of Tongji Medical College of Huazhong University of Science and Technology between May 2016 and March 2023 were analyzed, which included 31 patients with murine CAR-T and 49 with humanized products. Results: The proportion of patients with cytokine-release syndrome (CRS) in the murine and humanized groups was 63.1% and 65.3%, respectively. Moreover, a higher proportion of patients suffered from severe CRS in the murine group than in the humanized CAR-T group (19.4% vs 8.2%, P=0.174). Furthermore, one patient per group died of grade 5 CRS. The incidence of grade 1-2 immune effector cell-associated neurotoxicity syndrome (ICANS) was 12.9% and 6.1%, respectively; severe ICANS were not observed. Among patients receiving murine CAR-T-cells, an overall response (OR) was observed in 74.2%. Conversely, the OR rate of patients receiving humanized CAR-T-cells was 87.8%. During the median follow-up time of 10.5 months, the median recurrence-free survival (RFS) of patients with murine CAR-T-cells was 12 months, which was as long as that of patients with humanized CAR-T-cells. The median overall survival (OS) were not reached in both groups. Of the 45 patients with a bone marrow burden over 20% at baseline, humanized CAR-T therapy was associated with a significantly improved RFS (43.25% vs 33.33%, P=0.027). Bridging transplantation was an independent factor in prolonging OS (χ(2)=8.017, P=0.005) and PFS (χ(2)=6.584, P=0.010). Common risk factors, such as age, high proportion of bone marrow blasts, and BCR-ABL fusion gene expression, had no significant effect on patients' long-term follow-up outcomes. Three patients reached complete remission after reinfusion of humanized CAR-T-cells. However, one patient relapsed one month after his second infusion of murine CAR-T-cells. Conclusions: The results indicate that humanized CAR-T therapy showed durable efficacy in patients with a higher tumor burden in the bone marrow without any influence on safety. Moreover, it could overcome immunogenicity-induced CAR-T resistance, providing treatment options for patients who were not treated successfully with CAR-T therapies.

Deciphering Hypoplastic Myelodysplastic Syndrome and Aplastic Anemia via In-Depth Analysis of Lymphocyte Subsets

To explore the difference of lymphocyte subsets in peripheral blood (PB) between aplastic anemia (AA) and hypoplastic myelodysplastic syndrome (hypo-MDS) patients, meanwhile to compare the clinical parameters obtained from PB and bone marrow (BM). The lymphocyte subsets in hypo-MDS (n=25) and AA (n=33) patients were investigated by flow cytometry. Meanwhile, the differences in PB cell counts, biochemical indicators, BM cell counts and abnormal chromosomes between the two groups were analyzed. The percentage of CD8+T cells in AA group was significantly higher than that in hypo-MDS group (P=0.001), while the percentage of CD4+ T cells and the CD4+/CD8+ ratio in AA group were obviously lower than those in hypo-MDS group (P=0.015 and 0.001, respectively). Furthermore, the proportion of CD4+ and CD8+ activated T (TA) cells, and memory Tregs in AA group was distinctly lower than those in hypo-MDS group (P=0.043, 0.015 and 0.024, respectively). Nevertheless, the percentage of CD8+ naive T (TN) cells in AA patients was remarkably higher (P=0.044). And hypo-MDS patients had declined lymphocyte counts (P=0.025), increased levels of total bilirubin (TBil), lactate dehydrogenase (LDH), vitamin B12 and proportion of BM blasts than AA patients (P=0.019, 0.023, 0.027 and 0.045, respectively). In this study it was confirmed that the percentages of CD4+ and CD8+ TA cells, memory Tregs and CD8+ TN cells were significantly different between AA and hypo-MDS patients, which provide an essential basis for the identification of these two diseases.

Clinical Characteristics of Acute Myeloid Leukemia Patients with RUNX1 Gene Mutation

To investigate the incidence of Runt-related transcription factor 1 (RUNX1) gene and its associated gene mutations in patients with acute myeloid leukemia (AML), and analyze its clinical characteristics and prognosis. The genomic DNA-PCR method was used to detect the exon of RUNX1 gene, and the gene mutations were analyzed by genetic sequencing. NPM1, DNMT3A, FLT3-ITD, IDH1/2, K/N-RAS, CEPBA, TET2, and WT1 co-mutations were also detected. Patients were followed up to determine efficacy and prognosis. Among 171 patients, the RUNX1 gene mutation was detected in 17 cases, and the mutation rate was 9.9%. The type of RUNX1 gene mutation was 9 missense mutations, 4 frameshift mutations, and 4 nonsense mutations. The peripheral blood leukocyte count of the patients in mutation group was 3 (1-101) ×109/L, which was significantly lower than those in the non-mutation group [26 (1-298)×109/L] (P=0.002), while the platelet count was 79 (22-166)×109/L, which was higher than 50 (8-351)×109/L in the non-mutation group (P=0.010), and the proportion of bone marrow blasts of the patients in the mutation group was 37 (0-72)%, which was lower than 53 (0-98)% in the non-mutation group (P=0.020). The RUNX1 mutation rate in M0 type was 55.6%, and in M4 type was 13.6%, which was significantly higher than other FAB subtypes (P=0.003). There was no significant difference in age, sex, hemoglobin concentration, and counts of peripheral blood mononuclear cells (P>0.05). The prognosis of cytogenetics in the patients in the middle and high-risk groups was 88.1% and 89.7%, which were significantly higher than that in the low-risk group, and the difference showed statistically significant (P=0.018). There was no significantly relationship between RUNX1 and specific karyotype abnormalities, including Trisomy 8, Del (7q), t (8; 21), and Inv (16) (P>0.05). There was a significant relationship between RUNX1 gene mutation and IDH1/2, N/K-RAS gene mutation (P<0.01). The complete response rate (CR) of the patients with chemotherapy in the RUNX1 mutation group(37.5%) was significantly lower than 79.4% in the non-mutated group (P=0.001). The overall survival (OS) of the patients in RUNX1 gene mutation group was lower than that in non-mutation group (P<0.05). AML patients with RUNX1 gene mutation shows unique clinical and biological characteristics, RUNX1 mutation can be regarded as a molecular marker of poor prognosis in AML patients.

Reinduction chemotherapy regimen involved decitabine and cladribine improves the prognosis of patients with relapsed or refractory acute myeloid leukemia: A preliminary study.

Relapsed/refractory acute myeloid leukemia (R/R-AML) is characterized by a high incidence, short survival and poor prognosis. Presently, no unified effective reinduction chemotherapy regimen has been developed. Therefore, the use of reinduction chemotherapy regimens before allogeneic hematopoietic stem cell transplantation (allo-HSCT) is controversial. Our study aims to analyze the prognostic factors of R/R-AML and to evaluate the efficacy of the regimen involved decitabine, cladribine, idarubicin or homoharringtonine, and cytarabine (DCIA/DCHA). Clinical and survival data of 112 R/R-AML patients were obtained. Among the 102 R/R-AML patients that were treated with conventional regimens, we found that poor prognosis was related to a greater proportion of bone marrow blasts (>70%) and not achieving complete remission (non-CR) after the first reinduction chemotherapy. Hematopoietic stem cell transplantation (of which 89.47% was allo-HSCT) following CR after the first reinduction chemotherapy often improves the prognosis. Of the 10 R/R-AML patients that were treated with the DCIA/DCHA regimen, nine patients achieved CR or complete response with incomplete hematopoietic recovery (CRi) after one course of chemotherapy. The median overall survival of the 10 patients was 10.14 (1.23-29.13) months. In conclusion, non-CR was associated with poor prognosis in R/R-AML. Therefore, intensive reinduction chemotherapy should be selected to achieve CR. This creates conditions for allo-HSCT and improves prognosis of R/R-AML patients. The DCIA/DCHA regimen showed good efficacy and tolerable adverse reactions in R/R-AML treatment. This combination may be used as a bridging regimen for allo-HSCT in R/R-AML.

Relation of ASXL2 Gene Mutation with Clinical Characteristics, Prognosis and C-KIT Gene Mutation in AML Patients with AML1- ETO Fusion Gene

To analyze relation of ASXL2 gene mutation with the clinical characteristics, prognosis and C-KIT gene mutation in acute myeloid leukemia (AML) patients with AML1-ETO fusion gene. The clinical data of 63 primary AML patients with AML1-ETO fusion gene were collected and retrospectively analyzed. The mutation of ASXL2 gene was directly sequenced by PCR. The clinical characteristics, C-KIT mutation rate and prognosis were compared between the patients with ASXL2 gene mutation (group A) and non-mutation (group B). Among 63 patients, 8 (12.70%) cases of ASXL2 mutation gene was detected. Hemoglobin level in peripheral blood of patients in group A was significantly lower than that in group B (P＜0.01). There was no significant difference in sex, ages proportion of bone marrow blasts, lymph node enlargement, peripheral blood leukocytes count and platelets between the two groups (P＞0.05). The infiltration of central nervous system, liver and spleen was not found in both groups. The expression of CD33 in group A was significantly lower than that in group B (P＜0.05), but the results of other immunophenotype analysis were not significantly different between the two groups (P＞0.05). The remission rate and median survival time were not significantly different between two groups (P＞0.05). The detection rate of C-KIT gene mutation were not significantly different between group A and group B (P＞0.05). Among AML patients with AML1-ETO fusion gene, ASXL2 gene mutation accounts for a certain ratio, and the peripheral blood hemoglobin concentration and CD33 expression in these patients are often low. At the same time, ASXL2 gene mutation may not be closely related with C-KIT gene mutation.

The impact of KIT and other concomitant gene mutations on the prognoses of patients with core-binding factor acute myeloid leukemia

Objective: To study the impact of KIT and other concomitant gene mutations on the prognoses of patients with core-binding factor acute myeloid leukemia (CBF-AML). Methods: A total of 104 newly diagnosed patients with CBF-AML in Hebei Yanda Lu Daopei Hospital from January 2014 to February 2018 were analyzed, and high-throughput gene sequencing for the detection of mutations among 58 genes was executed. Also, the clinical features of KIT mutation-positive CBF-AML (KIT+CBF-AML) patients and the effects of other concomitant gene mutations on the prognoses of patients were also analyzed. Results: A total of 56 cases (53.85%) with KIT mutations were found in 104 CBF-AML patients. Among this, KIT D816 mutation was the most common (32 patients), followed by the N822 mutation (17 patients). Patients with KIT+CBF-AML have a higher proportion of bone marrow blasts at the time of diagnoses and are more likely to have sex chromosome loss. Among the 52 patients with KIT+CBF-AML who were followed up, the allogeneic hematopoietic stem cell transplantation (allo-HSCT) group had a higher overall survival rate (OS) than that of the chemotherapy group (88.9% vs 57.1%, χ(2)=6.076, P<0.05). The event-free survival (EFS) and OS of patients with KIT+CBF-AML with FLT3 mutation were both significantly lower than those of the FLT3 mutation-negative group (EFS: 40.0% vs 72.3%, χ(2)=6.557, P<0.05; OS: 60.0% vs 87.2%, χ(2)=8.305, P<0.05). The OS of the patient with TET2 mutation was lower than that of the TET2 mutation-negative group (50.0% vs 87.5%, χ(2)=4.130, P<0.05). Conclusion: Patients with KIT+CBF-AML with concomitant gene mutations, especially FLT3 and TET2, have poor prognoses, which can be improved by allo-HSCT.

TP53 Abnormality in Myelodysplastic Syndrome

TP53 (tumor suppressor gene P53), one of the most important tumor suppressor genes, plays an important role in cell cycle arrest, cell senescence, apoptosis, differentiation and metabolism. The TP53 gene is located on 17p13.1, its encoded product is the transcription factor P53 protein, which is known as the "the guardian of the genome". Alterations of TP53 include mutations and deletions and are generally associated with advanced stages of disease, insufficient therapy-response and poor prognosis. The main purpose of our study was to comprehensively analyze the TP53 mutation and 17p deletion in MDS in our single center. To better understand the relationship between TP53 abnormality and clinical phenotype, prognosis, leukemia transformation, therapeutic response of MDS. Next generation sequencing (NGS) method combining with cytogenetics analysis were used, 36 common related AML/MDS/MPN related genes such as TP53, TET2, WT1, ASXL1, U2AF1, RUNX1, etc were covered. According to the 2016 WHO classification and prognosis score system and from June 2011 to June 2017, 88 newly diagnosed MDS patients including 17 MDS-SLD, 32 MDS-MLD,6 MDS-RS,19 MDS-EB-1,11 MDS-EB-2 and 2 MDS-U, 1 5q- syndrome were enrolled. TP53 mutation/deletions were found in twenty-two (25%) of the 88 newly diagnosed MDS patients, among them,7 MDS-SLD, 4 MDS-MLD, 2 MDS-RS, 1 5q-, 6 MDS-EB-1 and 6 MDS-EB-2. TP53 mutation/deletions cases had a higher proportion of bone marrow blasts compared with TP53 negative cases (P=0.009), At the same time, TP53 positive were highly correlated with MDS-EB-2 subtype (P=0.025), complex karyotype (P&lt;0.001). Based on a median follow-up time of 21(1-267) months in all pts, 13 patients (14.8%) progressed to AML and pts with TP53 mutation/deletions tended to progress to AML (P=0.056) with a shorter OS (P=0.005) and PFS (P=0.001). NGS data of accompanying mutation in other classical leukemia genes shown that compound TP53 and U2AF1 mutations were significantly associated with disease progression. For TP53 mutation/deletion group (n=22), we further sequenced the TP53 status at multiple time point of pre and after DAC treatment, results shown that all patients had persistent TP53 positive status before and after treatment. In conclusion, our results indicate that in MDS TP53 mutation/deletions is highly correlated with MDS-EB-2 subtype, IPSS high-risk group, and complex karyotype. TP53 mutations that occur in the early stages of MDS may contribute to disease progression and leukemia transformation in conjunction with other molecular abnormalities. DAC improves outcomes in patients with TP53 mutation/deletion but may not clear TP53 mutations. Disclosures No relevant conflicts of interest to declare.

SRSF2 Mutations Identify a Distinct Subtype of Myeloid Neoplasm Across Myelodysplastic Syndromes and Acute Myeloid Leukemia

Splicing factors are the most frequently mutated genes in myeloid neoplasms with myelodysplasia, including myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and acute myeloid leukemia with myelodysplasia-related changes (AML-MDS). SF3B1 mutations was shown to be strongly associated with disease phenotype with ring sideroblasts, and SF3B1-mutant MDS has been proposed as a distinct disease subtype (Malcovati et al. Blood 2015). Conversely, SRSF2 has been found to be mutated in chronic myelomonocytic leukemia (CMML), and to be associated with multilineage dysplasia and excess blasts in MDS, as well as with clinico-pathological features of secondary AML (Linsdley et al. Blood 2015). In this study, we carried out a comprehensive mutation analysis in a well-annotated prospective cohort of 551 patients with myeloid neoplasms with myelodysplasia, and focused on clinical and molecular features of SRSF2-mutated patients with the aim to characterize patterns of co-occurring mutations and to identify genotype-phenotype correlations.A core panel of 54 genes recurrently mutated in myeloid neoplasms was analyzed using an Illumina HiSeq (Illumina Inc., CA, USA). Multivariable survival analyses were performed by means of Cox proportional hazards regression accounting for left censoring of the observation at the time of mutation assessment. Unsupervised hierarchical clustering analyses were performed in order to identify homogeneous groups of patients with respect to the variables of interest.Overall, 102 patients carrying SRSF2-mutation were identified, including 37 MDS (2 MDS with single lineage dysplasia, 9 with multilineage dysplasia, 6 with ring sideroblasts, 20 with excess blasts), 40 MDS/MPN (35 CMML, 5 MDS/MPN unclassified) and 25 AML-MDS. The median number of co-mutated genes was 2 (range 0 to 5); SRSF2 mutation was the unique mutation detected in 11 cases (5 AML-MDS, 4 MDS/MPN and 3 MDS). The most frequently co-mutated genes were TET2 (31/102 cases, 30%), ASXL1 (28%), IDH1/IDH2 (24%), RUNX1 (21%), STAG2 (14%) and CBL (10%). A significant association was found between co-mutation of TET2 and SRSF2 and myeloid neoplasm with monocytosis (P<.001), whereas STAG2 and IDH1/IDH2 mutations were significantly associated with MDS/AML-MDS phenotype (P=.044 and P=.001, respectively). We applied stepwise logistic regression, and confirmed that co-mutation of TET2 was a highly specific independent predictor of MDS/MPN phenotype within SRSF2-mutated myeloid neoplasms (OR=9.13, P<.001, positive predictive value: 0.85; specificity: 0.89).Then, we carried out uni- and multivariable Cox-regression analyses, and found that SRSF2-mutant MDS/MPN showed a significantly better prognosis compared to MDS (HR=.44, P=.008) and AML-MDS (HR=.47, P<.001). Conversely, having 20% or more bone marrow blasts or exceeding this threshold during follow-up did not affect survival of patients with SRSF2-mutated MDS or AML-MDS (HR=1.56, P=.15), suggesting that the proportion of bone marrow blasts does not recognize distinct subsets within SRSF2-mutated MDS and AML-MDS.Finally, we performed unsupervised hierarchical clustering analyses including current WHO classification criteria and gene mutations. This analysis identified one cluster including patients with MDS/MPN according to current WHO criteria, and a second cluster comprising patients with MDS or AML-MDS, in which a distinct subgroup including SRSF2-mutated patients was identified, irrespective of WHO bone marrow blast cut-offs.In conclusion, these results suggest that SRSF2 mutation is able to identify a distinct subset of myeloid neoplasms at the boundaries of MDS and AML, irrespective of current WHO classification criteria. Conversely, SRSF2-mutant MDS/MPN show different co-mutation patterns and clinical outcomes, supporting a classification in a separate category according to current WHO criteria. The recognition of a distinct entity with a unique molecular basis may significantly improve the diagnosis, classification and treatment of patients with these disorders. DisclosuresNo relevant conflicts of interest to declare.

Different Mutant Splicing Factors Cause Distinct Missplicing Events and Give Rise to Different Clinical Phenotypes in Myelodysplastic Syndromes

IntroductionSplicing factor (SF) mutations represent a novel class of driver mutations in myelodysplastic syndromes (MDS), where SF3B1 and SRSF2 are most frequently affected. Although abnormal RNA splicing is thought to play a central role in the pathogenic mechanism of mutated SFs, little is known about exact gene targets whose abnormal splicing is responsible for the pathogenesis of MDS.MethodsWe enrolled a total of 480 patients with MDS, for whom complete clinical and pathological data were available. RNA sequencing was performed for bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells from 215 MDS patients. Observed splicing junctions were compared between samples with and without each SF mutation. In SF-mutated cases, NMD could cause severe degradation of abnormal transcripts and obscure the effect of SF-mutants. To sensitively detect abnormal transcripts otherwise degraded by nonsense-mediated RNA decay (NMD), analysis was also performed on BM/MNCs from 7 patients and CD34+ bone marrow cells from 3 patients with or without inhibition of NMD by cycloheximide (CHX). Common mutations and copy number variations were also investigated using targeted sequencing.ResultsSF3B1 and SRSF2 mutations were associated with distinct clinical phenotypes and outcomes. SF3B1-mutated cases typically showed isolated erythroid dysplasia and high proportion of ring sideroblasts, whereas SRSF2 mutations correlated with a significantly higher incidence of myeloid and megakaryocyte dysplasia (P<.001), higher proportion of bone marrow blasts (P=.02) and lower degree of erythroid dysplasia and proportion of ring sideroblasts (P<.001). SF3B1- and SRSF2-mutated myeloid neoplasms were also associated with a significantly different outcome, SRSF2-mutated neoplasms having a significantly shorter survival (HR=5.35, P<.001).To explore the molecular basis of these distinct features in terms of splicing defects, RNA sequencing data from SF3B1-mutated (n = 68) and SRSF2-mutated (n = 39) BM/MNCs and CD34+ cells were compared with those without known SF mutations (n = 91) to detect splicing events significantly enriched in SF-mutated cells. In total 748 and 589 splicing events were enriched in SF3B1- and SRSF2-mutated samples. Among these, 203 (27%) of SF3B1-specific events were observed almost exclusively in SF3B1-mutated samples;193 (95%) were caused by misrecognition of 3' splice sites of which ~50% resulted in frameshift. In contrast, in SRSF2-mutated cases, the predominant defects were alternative exon usage, which accounted for for 80% of the abnormalities. However, the effect of mutant-SRSF2 on abnormal splicing was generally small, with 89% showing only <3× more abnormal transcripts in SRSF2-mutated. Similar results were obtained for BM/MNCs for both mutations.Splicing defects of both SF-mutations involved substantially different set of genes. Aberrant splicing enriched in SF3B1- and SRSF2-mutated samples involved 12 and 7 cancer-related genes defined by the Cancer Gene Census with no genes overlapped in between. Of special interest among these was EZH2, which showed 2 SRSF2-associated alternative exons with a premature termination codon. EZH2 transcripts having these exons are expected to be susceptible to NMD-mediated degradation. A similar defect was also detected in another component of the polycomb repressive complex 2 (PRC2), implicated in compromised function of PRC2 in SRSF2-mutated cases. On the other hand, 2 genes involved in mitochondrial heme synthesis were significantly affected in SF3B1-mutated samples. In addition, an additional gene engaged in heme synthesis, ABCB7, was identified from experiments using NMD inhibition to detect 'masked splicing'. ABCB7 is one of the causative genes for congenital sideroblastic anemia and uniformly showed reduced expression in SF3B1-mutated samples, most likely due to abnormal splicing.ConclusionSF3B1 and SRSF2 mutations have distinct impacts on clinical phenotypes and outcomes together with RNA splicing. SF3B1 mutations caused misrecognition of 3' splice sites, frequently resulting in truncated gene products and/or decreased expression due to NMD. SRSF2 mutations were characterized by modest but more widespread alterations in exon usage of genes including multiple components of PRC2. Our results provide insights into the pathogenesis of SF-mutated MDS. DisclosuresNo relevant conflicts of interest to declare.

Expressions of CD96 and CD123 in Bone Marrow Cells of Patients with Myelodysplastic Syndromes.

Our goal was to detect the expression of CD96 and CD123 in myelodysplastic syndromes (MDS) and their proliferation and differentiation characteristics. Forty-three patients with MDS, thirteen AML patients diagnosed in the Hematology Department of General Hospital of Tianjin Medical University, and sixteen healthy controls were enrolled in this study. The expressions of CD96 and CD123 on CD34+CD38- bone marrow cells (BMC) of these patients and controls were measured by FACS. CD114 (GCSFR), EPOR, and CD110 (TPOR) expression on their CD34+CD38-CD96+ and CD34+CD38-CD96- BMC and these cells' apoptosis (Annexin V) were also detected by FACS. MDS patients had a significantly higher proportion of CD34+CD38-/CD34+ BMC (22.72 ± 23.91%) than normal controls (8.63 ± 9.35%) (p < 0.01). The expressions of CD34+CD38-CD96+/CD34+CD38- BMC and CD34+ CD38-CD123+/CD34+CD38- BMC of MDS patients (21.18 ± 24.06%, 31.62 ± 28.42%, respectively) were significantly higher than those of normal controls (11.23 ± 17.91%, 9.29 ± 12.63%) (p < 0.01). The expressions of CD34+CD38-CD96+/CD34+CD38- BMC and CD34+CD38-CD123+/CD34+CD38- BMC in the high-risk MDS group (26.25 ± 27.64%, 37.56 ± 31.01%) was significantly higher than those in the low-risk group (11.44 ± 10.88, 15.53 ± 10.96%) (p < 0.05). The expression levels of CD123 and CD96 were significantly correlated with the proportion of bone marrow blasts (r = 0.384, p < 0.05; r = 0.625, p < 0.05). The expressions of CD114, CD110, and Annexin V in CD34+CD38-CD96+ BMC (7.23 ± 10.25%, 0.52 ± 1.23%, 2.96 ± 3.84%, respectively) were significantly lower than those in CD34+CD38-CD96- BMC (24.35 ± 21.74%, 12.03 ± 10.91%, 13.69 ± 17.98%, respectively) of MDS patients (p < 0.05). There was a higher proportion of CD34+CD38-CD96+ and CD34+CD38-CD123+ cells in bone marrow of MDS patients. Those cells expressed lower cytokine receptors and displayed lower apoptosis. Leukemia stem cells (LSC) in MDS might be contained in those cells.

Clinical and genetic predictors of prognosis in myelodysplastic syndromes.

Myelodysplastic syndromes are a collection of clonal hematopoietic disorders with a wide range of clinical manifestations and eventual outcomes. Accurate prediction of a patient's prognosis is useful to define the risk posed by the disease and which treatment options are most appropriate. Several models have been created to help predict the prognosis for patients with myelodysplastic syndromes. The International Prognostic Scoring System (IPSS) has been the standard tool used to risk stratify MDS patients since its publication in 1997. Other models have since been created to improve upon the IPSS, including the recent Revised International Prognostic Scoring System. Most models include the presence or severity of peripheral blood cytopenias, the proportion of bone marrow blasts, and specific karyotype abnormalities. Other factors including age, performance status, co-morbidities, transfusion dependence, and molecular biomarkers can further refine the prediction of prognosis in some models. Novel, disease specific biomarkers with prognostic value in myelodysplastic syndromes including cell surface markers, gene expression profiles, and high resolution copy number analyses have been proposed but not yet adopted clinically. Somatic abnormalities in recurrently mutated genes are the most informative prognostic biomarkers not currently considered by clinical risk models. Mutations in specific genes have independent prognostic significance and, unlike cytogenetic abnormalities, are present in the majority of myelodysplastic syndrome cases. However, mutational information can be complex and there are challenges to its clinical implementation. Despite these limitations, DNA sequencing can refine the prediction of prognosis for myelodysplastic syndrome patients and has become increasingly available in the clinic where it will help improve the care of patients with myelodysplastic syndromes.

Expression of DLK1 Gene in the Bone Marrow Cells of Patients with Myelodysplastic Syndromes and Its Clinical Significance.

ObjectiveThis study aims to investigate the expression of delta-like 1 (DLK1) gene in the bone marrow cells of patients with myelodysplastic syndromes (MDS) and to explore its molecular characteristics for the early diagnosis of MDS.MethodsThe expression of DLK1 mRNA in the bone marrow cells of cases with MDS, acute myeloid leukemia (AML), and normal control groups were measured by real-time polymerase chain reaction and were analyzed for clinical significance.ResultsSignificantly higher expression of DLK1 mRNA was observed in the bone marrow cells of MDS patients (0.7342±0.3652) compared with the normal control group (0.4801±0.1759) (P<0.05). The expression of DLK1 mRNA had a positive correlation with the proportion of bone marrow blasts (r=0.467, P<0.05). Moreover, DLK1 mRNA expression was significantly increased as MDS progressed (P<0.05). Patients with abnormal karyotypes exhibited significantly higher expression of DLK1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Subsequently, patients with highly expressed DLK1 (≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower DLK1 expression (<0.8),(0.1517±0.3109), (P<0.05).ConclusionsThe DLK1 gene was highly expressed in MDS patients, and was increased as MDS progressed. The expression of DLK1 mRNA was positively correlated with the proportion of the bone marrow blasts. A high expression of DLK1 gene suggested a higher malignant clone burden of MDS.

TET2 Expression in Bone Marrow Mononuclear Cells of Patients with Myelodysplastic Syndromes and Its Clinical Significances.

ObjectiveTo investigate the expression of TET2 mRNA and protein in the bone marrow mononuclear cells (BMMNC) of patients with myelodysplastic syndrome (MDS) and its clinical significance.MethodsThe expression of TET2 mRNA and protein in bone marrow mononuclear cells (BMMNC) of 32 patients with MDS and 20 healthy donors was examined by qPCR and Western blot.ResultsThe expression of TET2 mRNA in BMMNC was down-regulated in MDS patients compared with the donor group [(0.41±0.28)% vs. (1.07±0.56)%] (P<0.001). Compared with lower expression group (TET2<0.4) [(6.53±6.17)%], patients with higher expression of TET2 (≥0.4) presented significantly lower proportion of bone marrow blasts [(1.21±1.56)%] (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=-0.412, P<0.05). The expression of TET2 protein was down-regulated in MDS patients compared with that in the donor group.ConclusionsThe mRNA and protein expression of TET2 in BMMNC of MDS patients is decreased, which might be useful as an important parameter for the evaluation of MDS clone burden.

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

Abstract 5042 ObjectiveTo 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. MethodsThe 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. ResultsThe 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). Conclusiondlk1 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.

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

Abstract 5036 ObjectiveThis 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. MethodsThe 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). ConclusionsThe 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.

Expression of CD123 and CD114 on the Bone Marrow Cells of Patients with Myelodysplastic Syndromes

AbstractAbstract 4966 BackgroundRecent studies have shown that interleukin-3 receptor α(CD123) is highly expressed on leukemia stem cells of patients with acute myeloid leukemia, and is correlated with tumor load and poor prognosis. The expression of CD123 may also be high in patients with myelodysplastic syndromes (MDS). In this study, the expression of CD123 as well as granulocyte colony stimulating factor (G-CSF) receptor (CD114) on the bone marrow cells of patients with MDS was investigated in order to explore the molecular marker of the malignant clone of MDS. MethodsForty-two patients with MDS, who were diagnosed in the hematological department of General Hospital of Tianjin Medical University from 2008 to 2009, and twelve normal controls were enrolled in this study. FACS was used to measure the expression of CD123 on CD34+CD38- cells and CD114 on CD34+ cells of the bone marrow of these patients and controls and the clinical significance was analyzed. The expression of CD114 on CD123+CD34+CD38- cells was further measured to explore the molecular marker of the malignant clone in MDS. ResultsThe ratio of CD34+CD38-/CD34+ in the bone marrow cells of MDS patients was [(14.03±5.27)%], significantly higher than that of normal controls [(7.70±4.36)%] (P<0.01); The ratio of CD123+CD34+CD38-/CD34+CD38- in the bone marrow cells of MDS patients[(48.39±28.15)%]was significantly higher than that of normal controls [(8.75±11.71)%] (P<0.01), and was significantly positively correlated with the proportion of bone marrow blasts(r=0.457, P<0.05). The ratio of CD114+CD34+/CD34+ in the bone marrow cells of MDS patients [(33.05±21.71)%] was lower than that of normal controls [(38.99±19.07)%], but with no significance(P>0.05). The expression of CD114 on CD123+CD34+CD38-cells [(34.82±29.58)%] was significantly lower than that on CD123-CD34+CD38-cells [(53.48±27.41)%] of MDS patients (P<0.05). ConclusionsMDS patients displayed higher proportion of CD34+CD38-/CD34+ than normal controls. CD123 was highly expressed in the bone marrow of patients with MDS, significantly correlated with the proportion of bone marrow blasts, thus might be the marker of MDS malignant clone. CD123+CD34+CD38-cells exhibited lower expression of G-CSF receptors, which might partly explain why MDS clone responsed worse to G-CSF in vitro and in vivo. Disclosures:No relevant conflicts of interest to declare.

Impact of adjunct cytogenetic abnormalities for prognostic stratification in patients with myelodysplastic syndrome and deletion 5q.

This cooperative study assessed prognostic factors for overall survival (OS) and risk of transformation to acute myeloid leukemia (AML) in 541 patients with de novo myelodysplastic syndrome (MDS) and deletion 5q. Additional chromosomal abnormalities were strongly related to different patients' characteristics. In multivariate analysis, the most important predictors of both OS and AML transformation risk were number of chromosomal abnormalities (P<0.001 for both outcomes), platelet count (P<0.001 and P=0.001, respectively) and proportion of bone marrow blasts (P<0.001 and P=0.016, respectively). The number of chromosomal abnormalities defined three risk categories for AML transformation (del(5q), del(5q)+1 and del(5q)+ ≥ 2 abnormalities) and two for OS (one group: del(5q) and del(5q)+1; and del(5q)+ ≥ 2 abnormalities, as the other one); with a median survival time of 58.0 and 6.8 months, respectively. Platelet count (P=0.001) and age (P=0.034) predicted OS in patients with '5q-syndrome'. This study demonstrates the importance of additional chromosomal abnormalities in MDS patients with deletion 5q, challenges the current '5q-syndrome' definition and constitutes a useful reference series to properly analyze the results of clinical trials in these patients.

Chronic Myelomonocytic Leukemia (CMML) with More Than 15% of Ring Sideroblasts in Bone Marrow: An Overlapping Disorder Between CMML and Refractory Anemia with Ring Sideroblasts.

Abstract 290The main diagnostic criteria for chronic myelomonocytic leukemia (CMML), a heterogeneous disorder sharing features of myelodysplastic syndromes (MDS) and chronic myeloproliferative disorders, is the existence of a sustained absolute monocyte count in peripheral blood (PB) above 1 × 109/L. On the other hand, the presence of more than 15% ring sideroblasts (RS) in bone marrow (BM) is a well recognized morphological feature of dyserithropoiesis and, in the absence of blasts in PB and less than 5% blasts in BM, is diagnostic of refractory anemia with ring sideroblasts (RARS) with or without multilineage dysplasia. In FAB as well as in WHO classification systems for myeloid neoplasms those cases presenting with both an absolute monocyte count in PB above 1 × 109/L and more than 15% RS in BM are diagnosed of CMML but the preeminence given to the monocyte count in PB over the proportion of RS in BM is not evidence-based. The main purpose of this study was to assess the clinical and biological characteristics and outcome [overall survival (OS) and acute leukemic (AL) evolution] of a series of 77 patients diagnosed of CMML by FAB and WHO criteria who had more than 15% RS in BM at presentation (CMML-RS) and to compare them with those of a series of 417 patients with CMML with less than 15% RS (classical CMML) and those of a series of 178 patients with classical RARS (38 patients with and 140 patients without multilineage dysplasia). Comparisons of proportions and ranks of variables between different groups were performed by chi square or Mann-Whitney-U tests as appropriate. Actuarial curves of OS and risk of AL evolution were built by Kaplan-Meier method and differences between curves compared with log-rank tests. Multivariate analyses of OS and risk of AL evolution were performed by Cox proportional hazards regression method. Patients with CMML-RS had lower hemoglobin level (P=0.008), lower absolute counts of leukocytes (P<0.001), neutrophils (P=0.002), and monocytes (P<0.001), higher platelet count (P<0.001), lower proportion of blasts in PB (P=0.015) and BM (P=0.035), and higher serum level of ferritin (P<0.001) and LDH (P=0.06) than patients with classical CMML. Patients with CMML-RS had significantly better OS than patients with classical CMML (median, 79 mo and 26 mo respectively; P<0.001; Figure) as well as lower risk of AL evolution (cumulative proportion at 5 yr, 7% and 20% respectively; P=0.07). Further, the beneficial prognostic relevance of the proportion of RS in BM on OS was maintained in multivariate analyses (P<0.001). In marked contrast, OS (median, 64 mo; Figure) and risk of AL evolution (cumulative proportion at 5 yr, 9%) of patients with classical RARS were closely similar to those observed in patients with CMML-RS (P>0.90). Patients with classical RARS were more anemic (P=0.001), had lower absolute counts of leukocytes (P<0.001), neutrophils (P=0.01), and monocytes (P<0.001), higher platelet count (P=0.002), lower proportion of blasts in PB (P=0.01) and BM (P<0.001), and lower serum level of ferritin (P=0.01) and LDH (P=0.11) than patients with CMML-RS. To avoid the potential interference in the analyses of disparities in the proportion of blasts in BM in the different groups of patients all the analyses were repeated excluding from all the groups those cases with 5% or more blasts in BM. Fifty-three patients with CMML-RS, 245 with classical CMML, and all 178 with classical RARS were evaluable for these sub-analyses. The results obtained were similar to those in the overall series of patients (data not showed). To sum up, all these results show that the proportion of RS in BM is a much powerful prognostic indicator than absolute monocyte count in PB in CMML and demonstrate that the presence of a proportion of RS greater than 15% in BM in patients with CMML defines a subset of patients that clearly differ in their biological characteristics from classical CMML and classical RARS. CMML-RS has a clinical course very close to that of classical RARS and markedly better than classical CMML. These data strongly suggest that CMML-RS is an overlapping syndrome between CMML and RARS. For clinical purposes patients with >1 × 109 monocytes/L in PB and >15% RS in BM should be better classified as RARS than as CMML. The WHO classification needs to be revisited to account for those findings. [Display omitted] Disclosures:No relevant conflicts of interest to declare.