Qian Sx

Overexpression of miR-378 is frequent and may affect treatment outcomes in patients with acute myeloid leukemia

MicroRNA miR-378 plays important roles in tumorigenesis by enhancing cell survival, reducing apoptosis, promoting tumor growth, angiogenesis and promoting cell migration and invasion. Abnormal expression of miR-378 has been observed in various types of cancers. The aim of this study was to investigate the expression status of miR-378 and its clinical significance in patients with acute myeloid leukemia (AML) using real-time quantitative PCR. miR-378 overexpression was identified in 26 of 84 (31%) AML patients. The patients with miR-378 overexpression had lower hemoglobin level than those without miR-378 overexpression (66 versus 78 g/L, respectively, P=0.010). The frequency of miR-378 overexpression in FAB-M2 subtype was higher than other subtypes (44% versus 20%, P=0.032). Moreover, the frequency of miR-378 overexpression was higher in patients with t(8;21) than in others (64% versus 24%, P=0.012). The status of miR-378 expression was not correlated with the mutations of eight genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, C/EBPA and U2AF1). The difference in relapse-free survival was observed between patients with and without miR-378 overexpression (P=0.049). These findings suggest that miR-378 up-regulation is a common event and might have an adverse impact on prognosis in AML.

Cotransplantation of HLA-identical mesenchymal stem cells and hematopoietic stem cells in Chinese patients with hematologic diseases

Mesenchymal stem cells (MSCs) may be employed to support hematopoietic reconstitution and mitigate graft‐vs.‐host disease (GVHD) in transplantation of hematopoietic stem cells (HSCs). The aim of this study was to explore the feasibility and safety of cotransplantation culture‐expanded MSCs and HSCs from the same human leukocyte antigen (HLA)‐identical sibling donor in Chinese patients with hematologic diseases. Bone marrow mononuclear cells from healthy donors were cultured and expanded ex vivo. Immunophenotype, adipogenic and osteogenic differentiation potential, and karyotype of the harvested MSCs were detected on those who had been cotransplanted with HSCs and MSCs from the same donor. Hematopoietic reconstitutions, complications, and clinical outcomes were observed after cotransplantation in these patients. (1.77 ± 0.40) × 106/kg (donor’s weight) MSCs were successfully expanded from 23.6 ± 5.96 ml of bone marrow samples. They had normal karyotypes with bi‐lineages differentiation potential, and were CD73, CD90, and CD105 positive. Twelve patients underwent cotransplantation with no observable adverse response during and after the infusion of MSCs. Hematopoietic reconstitutions were rapid. Two patients developed grade II–IV acute GVHD, and two extensive chronic GVHD. Four patients suffered from cytomegalovirus infection but were cured eventually. Up to now, seven patients have been followed as long as 29–57 months and five patients died. It is concluded that MSCs can be expanded effectively by culture and it is safe and feasible to cotransplant patients with allogenic culture‐expanded MSCs and HSCs.

Clinical importance of different calreticulin gene mutation types in wild-type JAK2 essential thrombocythemia and myelofibrosis patients.

The Janus kinase 2 (JAK2) V617F mutation (JAK2 V617F), JAK2 exon 12 mutations and myeloproliferative leukemia virus oncogene W515L/K mutation (MPL W515L/K) have become three major molecular diagnosis criteria for myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) from 2005.1 However, diagnosing MPNs with non-mutated JAK2 and MPL remains a major diagnostic challenge.2–4 Some recent studies have reported calreticulin (CALR) gene mutations in patients with non-mutated JAK2 V617F MPNs.5–7 There are some distinct mutation types in MPN subtypes, but the differences in the clinical significance and prognosis among the different mutation types are obscure.8–10 Here, we report our data on CALR mutation in wild-type (wt) JAK2 MPN on patients. It should also be mentioned that this is undoubtedly the first report regarding CALR mutations in Chinese MPN patients. From January 2008 to December 2013, bone marrow or peripheral blood samples from 301 MPNs patients were collected in the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province, China, including ET (n=222), PV (n=37), PMF (n=33), post-ET MF (PET-MF; n=6), and post-PV MF (PPV-MF; n=3). We also obtained bone marrow samples from 174 patients with other myeloid neoplasms including: myelodysplastic syndrome (MDS; n=8), chronic myelogeneous leukemia (CML; n=55), acute myeloid leukemia (AML; n=104), and chronic myelomonocytic leukemia (CMML; n=7), as well as peripheral blood samples from 121 healthy controls. All participants provided their informed consent. Genomic PCR combined with direct and cloning sequencing was applied to screen CALR mutations. A total of 24.3% (73 of 301) patients with MPNs were found harboring CALR mutations. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of patients with ET and PMF, respectively (Figure 1A). Moreover, CALR mutations were found in 57.0% (69 of 121) ET patients with wt JAK2 and 30.8% (4 of 13) PMF patients with wt JAK2. No CALR mutation in patients with PV, PET-MF, PPV-MF (Figure 1A) was found. The CALR mutations have multiple deletions or insertions including: L367fs*46 (33 of 74; 44.6%), K385fs*47 (25 of 74; 33.8%), K368fs*51 (3 of 74; 4.1%), Q365fs*50 (3 of 74; 4.1%), E364fs*49 (2 of 74; 2.7%), K374fs*56 (2 of 74; 2.7%), L367fs*48 (1 of 74; 1.4%), Q365fs*48 (1 of 74; 1.4%), E364fs*55 (1 of 74; 1.4%), K375fs*48 (1 of 74; 1.4%), K375fs*55 (1 of 74; 1.4%), and K377fs*50 (1 of 74; 1.4%). Figure 1. (A) Frequency of CALR mutations in myeloid neoplasms and healthy control. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of ET and PMF patients, respectively. Approximately 1% of patients (1 of 104) with AML were found to harbor ... These patients with MPNs were simultaneously examined for the presence of other gene mutations. PV patients were screened for JAK2 V617F and JAK2 exon 12 mutations, while ET and MF patients were screened for JAK2 V617F and MPL W515L/K mutation. Among the total 301 patients with MPNs, 52.2% (157 of 301) were found to harbor JAK2 V617F mutation. Among the 222 patients with ET and 37 patients with PV, 0.9% (2 of 222) were found to harbor MPL W515L/K mutations and 2.7% (1 of 37) to harbor JAK2 exon 12 mutation, respectively (Figure 1B). JAK2 V617F, JAK2 exon 12 mutation, MPL W515L/K mutations and CALR mutations were found exclusively in these MPNs patients. We also screened CALR mutations in 104 AML patients, 55 CML patients, 7 CMML patients, and 8 MDS patients (including 5 refractory cytopenia with multilineage dysplasis, 2 refractory anemia with excess blasts, and one refractory anemia) to investigate whether CALR mutations were present in other myeloid neoplasms. Although most of these patients had negative results, one AML patient (59-years old, male, M2 subtype) was found to harbor CALR mutation (L367fs*46) without JAK2 V617F and MPL W515L/K mutations (Figure 1A). This patient had no previous history of MPN or MDS, Fms-related tyrosine kinase 3 internal tandem duplication, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog mutation, nucleophosmin mutation. CCAAT/enhancer binding protein alpha mutation was all negative and cytogenetics analysis showed normal karyotype. In addition, no CALR mutation was detected in the 121 healthy controls (Figure 1A). For mutation types, a total of 12 distinct variants of CALR mutation, including 11 deletions and one insertion, were identified in our patients. L367fs*46, which resulted from a 52-bp deletion, and K385fs*47, which resulted from a 5-bp insertion, were the most frequent CALR mutations. The two mutations accounted for 44.6% (33 of 74) and 33.8% (25 of 74) in all cases with mutant CALR, respectively. For ET patients, the two mutations were 14.0% (31 of 222) and 10.4% (23 of 222), respectively. For PMF patients, the two mutations were 3.0% (1 of 33) and 6.1% (2 of 33), respectively. There was no significant difference in the two mutation types between patients with ET and PMF (P=0.137 and P=0.645, respectively). Moreover, the two mutations were 44.9% (31 of 69) and 33.3% (23 of 69) in CALR mutation positive ET patients (Figure 1C), as well as 25% (1 of 4) and 50% (2 of 4) in CALR mutation positive PMF patients (P=0.793 and P=0.888, respectively). There were few other mutation types in the CALR-mutated samples (Figure 1C). ET patients with mutant CALR were significantly younger (P<0.001) and had lower white blood cell (WBC) counts (P<0.001), lower hemoglobin (Hb) levels (P=0.002), and higher platelet (PLT) counts (P<0.001) than patients with JAK2 V617F. No significant difference can be identified between ET patients with mutant CALR and JAK2 V617F in terms of sex and thrombotic events (Table 1). Similarly, PMF patients with mutant CALR showed lower Hb level (P=0.001) than JAK2 V617F. There was no significant difference in sex, age, WBC count, PLT counts or thrombotic events between PMF patients with mutant CALR and JAK2 V617F (Table 1). For different CALR mutations in ET patients, younger age (P=0.020), lower WBC count (P<0.001), and lower Hb level (P=0.002) were observed in CALR L367fs*46 than JAK2 V617F. In addition, ET patients with CALR K385fs*47 showed lower age (P<0.001), lower WBC counts (P<0.001), lower Hb levels (P=0.025) and higher PLT counts (P=0.005) than JAK2 V617F (Table 2). Table 1. Clinical features of essential thrombocythemia and primary myelofibrosis patients with CALR and JAK2 mutation. Table 2. Clinical characteristics of essential thrombocythemia patients with different types of CALR and JAK2 mutation. The overall survival (OS) rates of patients with ET and PMF were analyzed using the Kaplan-Meier curve. Longer OS was observed in ET and PMF patients with mutant CALR, but not wt CALR (P=0.511 and P=0.729, respectively) (Table 1 and Figure 1D). According to the risk stratification system in ET,11 there was a significant difference between patients with ET in the CALR-mutated group and JAK2 V617F mutant group or wt CALR group (both P<0.001) (Table 1). In summary, our data from this large cohort of Chinese patients with MPNs confirmed CALR mutations were novel molecular markers in wt JAK2 MPNs. It should always be noted that the combination of CALR, JAK2, and MPL W515L/K mutation analysis could contribute to the diagnosis of MPNs.5–7 Different CALR mutations of patients with MPNs had distinct clinical characteristics. Patients with the L367fs*46 and K385fs*47 mutations have shown a favorable prognosis, but further research is required to confirm this result. Given the relative proportion of MPN patients without JAK2/MPL/CALR mutations in our patient group, further investigation should be carried out to find novel molecular aberrations.

Clinical significance of lymphoid enhancer-binding factor 1 expression in acute myeloid leukemia.

Abstract Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/β-catenin signaling pathway and its dysregulation is associated with a number of malignant diseases such as leukemia. We explored the expression profile of LEF1 in acute myeloid leukemia (AML) and determined its specific prognostic significance in this disease. The LEF1 mRNA level in patients with previously untreated AML was significantly higher than in normal controls. Patients with AML with relatively higher LEF1 expression were more likely to achieve a complete remission (CR) following induction therapy in comparison to those with a lower LEF1 level. Moreover, we provide the first evidence that primary AML samples with AML1–ETO or PML–RARα have a higher LEF1 level compared with those without each fusion gene. High LEF1 expression predicts a significantly better overall survival for patients with intermediate-risk cytogenetics. High LEF1 level was associated with a favorable relapse-free survival in patients with FLT3-ITD wild-type. Finally, a scoring system based on LEF1 level and mutation status of FLT3-ITD or NPM1 is reliable to predict the outcome for AML with intermediate-risk cytogenetics. Our results indicate that LEF1 contributes to the pathophysiology of AML and could serve as a novel predictor of better treatment response. LEF1 level may be incorporated into an improved risk classification system for certain specific subtypes of AML.

The prognostic evaluation of CLLU1 expression levels in 50 Chinese patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the relentless accumulation of monoclonal B cells with the appearance of small mature lymphocytes and a characteristic CD5 and CD19 co-expression immunophenotype. The incidence of CLL is lower in Asian countries than in Western countries, where CLL is the most common leukemia. To investigate CLLU1 expression in CLL and explore the relationship between CLLU1 expression and alternative prognostic markers, we measured CLLU1 expression levels by semiquantitative RT-PCR in a cohort of 50 Chinese patients with CLL. Analyses of IgVH somatic mutational status, ZAP-70 expression, CD38 expression, and chromosomal aberrations were also performed. The expression of CLLU1 mRNA was determined in 26 of the 50 cases (52%), among which 7 at Binet A (7/21, 33.33%) and 19 at Binet B + C (19/29, 65.52%). The expression levels of CLLU1 were significantly increased in B + C CLL patients at Binet stage compared with those at Binet stage A (P = 0.005). Data for the IgVH somatic mutational status were available for 20 patients with known CLLU1 expression. Five (25%) patients, all expressed CLLU1 mRNA, displayed unmutated IgVH gene usage. While in 15 patients (15/20, 75%) with mutated IgVH gene, only 6 were CLLU1 positive. Patients with unmutated IgVH genes expressed higher levels of CLLU1 than did those with IgVH mutations (P < 0.05). Among 24 CD38+-CLL cases, 17 (70.83%) were CLLU1 positive, whereas only 9 (34.62%) positive cases were identified in 26 CD38−-CLL patients. Thus, the expression of CLLU1 in CD38+-CLL was significantly higher than that in CD38−-CLL. However, no significant difference of CLLU1 expression was found between ZAP-70+ (14/22, 63.64%) and ZAP-70− (12/28, 42.86%) patients (P > 0.05). We conclude that CLLU1 expression was significantly associated with clinical stages, IgVH somatic mutational status and CD38 expression, and might be an important prognostic factor in CLL patients.

TIGAR cooperated with glycolysis to inhibit the apoptosis of leukemia cells and associated with poor prognosis in patients with cytogenetically normal acute myeloid leukemia

BackgroundCancer cells show increased glycolysis and take advantage of this metabolic pathway to generate ATP. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects tumor cells from intracellular reactive oxygen species (ROS)-associated apoptosis. However, the function of TIGAR in glycolysis and survival of acute myeloid leukemia cells remains unclear.MethodsWe analyzed TIGAR expression in cytogenetically normal (CN-) AML patients and the correlations with clinical and biological parameters. In vivo and in vitro, we tested whether glycolysis may induce TIGAR expression and evaluated the combination effect of glycolysis inhibitor and TIGAR knockdown on human leukemia cell proliferation.ResultsHigh TIGAR expression was an independent predictor of poor survival and high incidence of relapse in adult patients with CN-AML. TIGAR also showed high expression in multiple human leukemia cell lines and knockdown of TIGAR activated glycolysis through PFKFB3 upregulation in human leukemia cells. Knockdown of TIGAR inhibited the proliferation of human leukemia cells and sensitized leukemia cells to glycolysis inhibitor both in vitro and in vivo. Furthermore, TIGAR knockdown in combination with glycolysis inhibitor 2-DG led leukemia cells to apoptosis. In addition, the p53 activator Nutlin-3α showed a significant combinational effect with TIGAR knockdown in leukemia cells. However, TIGAR expression and its anti-apoptotic effects were uncoupled from overexpression of exogenous p53 in leukemia cells.ConclusionsTIGAR might be a predictor of poor survival and high incidence of relapse in AML patients, and the combination of TIGAR inhibitors with anti-glycolytic agents may be novel therapies for the future clinical use in AML patients.

Heterogeneous leukemic clones identified by NPM1 mutation analysis in patient with acute monocytic leukemia

Abstract NPM1 mutation is the most common molecular abnormality in patients with acute myeloid leukemia (AML), especially normal karyotype AML (NK-AML), and is associated with a favorable prognosis in the absence of concomitant FLT3-ITD. Like other molecular abnormalities such as FLT3-ITD, C/EBPα and c-Kit mutation, NPM1 mutation normally presents as a recurrent molecular abnormality. The NPM1 mutation is generally used as a molecular marker in the prognosis evaluation of a patient with AML. Here, we report a different case. He was first diagnosed with NPM1 mutation-positive acute monocytic leukemia. However, he achieved no remission, but the NPM1 mutation dramatically became negative after induction chemotherapy. Finally, he achieved complete remission after salvage chemotherapy and the NPM1 mutation was still negative. To our knowledge, this is a rare case according to the worldwide published literature.

A Case of Ulcerative Colitis Associated With Autoimmune Hemolytic Anemia Successfully Treated by Autologous Hematopoietic Stem Cell Transplantation

A Case of Ulcerative Colitis Associated With Autoimmune Hemolytic Anemia Successfully Treated by Autologous Hematopoietic Stem Cell Transplantation

The combination therapy of imatinib and dasatinib achieves longterm molecular response in two imatinib-resistant and dasatinibintolerant patients with advanced chronic myeloid leukemia.

For patients with chronic myeloid leukemia (CML) failing imatinib therapy, second-generation tyrosine kinase inhibitors (TKIs) are recommended. Here, we describe two patients with advanced CML who failed imatinib therapy and did not tolerate the recommended dose of dasatinib, but then achieved a major molecular response with the combination of imatinib and dasatinib with no significant extramedullary toxicity. Our observations suggest that combination of TKIs may provide an additive/synergistic antileukemic effect.

The synergy of Vitamin C with decitabine activates TET2 in leukemic cells and significantly improves overall survival in elderly patients with acute myeloid leukemia

BACKGROUND Decitabine is widely used in the treatment of acute myeloid leukemia (AML) in elderly patients. Low-dose Vitamin C has also been indicated to induce DNA demethylation at the cellular level. However, little is known whether low-dose Vitamin C has a synergistic effect with decitabine in clinic. METHODS The effect of combined low-dose Vitamin C and decitabine on cell proliferation, the cell cycle, apoptosis and the expression level and activity of TET2 was investigated in HL60 and NB4 human leukemic cells. Additionally, we analyzed the clinical outcomes of 73 elderly AML patients who received A-DCAG (intravenous Vitamin C [IVC] plus DCAG [n = 39]) or DCAG (n = 34) treatment. RESULTS We found that low-dose Vitamin C and decitabine has a synergistic efficacy on proliferation, apoptosis, TET2 expression and activity, compared to drug-alone treatment in HL60 and NB4 cell lines in vitro. In clinic, feasibility and safety evaluations revealed that patients who received A-DCAG regimen have a higher complete remission (CR) rate than those who received the DCAG regimen (79.92% vs. 44.11%; P = 0.004) after one cycle of chemotherapy. The median overall survival (OS) was better in the A-DCAG group compared with the DCAG group (15.3 months vs. 9.3 months, P = 0.039). Patients with adverse cytogenetics did benefit from CR. There was no clinically significant additional toxicity observed with the addition of IVC. CONCLUSION On the basis of these results, the addition of IVC at low doses to DCAG appeared to improve CR and prolong OS, compared with DCAG, in elderly patients with AML.