| Objective:Acute myeloid leukemia (AML) is a large family of highly heterogeneous hematological malignancies. t (8; 21) (q22, q22) is one of the most frequent chromosomal translocations occurring in AML, and the fusion gene AML1-ETO is considered as a significant characteristics and leukemia-initiating event in t (8; 21) AML. A lot of researches confirmed that alternative splicing variants AMLl-ET09a are closely related with occurrence, development and prognosisi of t (8; 21) AML. However, it is not clear what the role the regulation mechanism played in producing of alternative splicing variants AML1-ETO9a.Materials and methods:1.21 cases of AML 1-ETO positive paired samples (obtained from the same patient in different courses, including initial treatment or relapse and response to treatment) and two AML1-ETO negative (also from AML-M2 patient) paired samples were collected for high-throughput sequencing of exon to analyze the number of somatic mutations. A mutation reproducible analysis of 23 samples was carried out; 2. The overall expression level of serine/arginine-rich splicing factor(SRSF) in different karyotype samples was analyzed based on a large number of AML clinical samples and normal bone marrow cDNA microarray database; 22AML1-ETO-positive cases, 22 AMLl-ETO-negative cases and 5 normal human bone marrow cases from cDNA microarray database were selected to identify splicing factors, SRSF2, SRSF6 and SRSF11, which were expressed significantly higher in AML1-ETO positive samples; a signaling pathway analysis was carried out on 145 proteins with significant difference via cloning of ETO9a exons and their flanking sequences, in vitro transcription, RNA precipitation (RIP) and mass spectrometry; the results of RIP and mass spectrometry were verified with Western blot; 3. The effects of SRSF2 on alternative splice variant AML1-ET09a as well as AML1-ETO negative and positive cell proliferation and malignant phenotype were confirmed by creating SRSF2 lentivirual expression vector, transfection of cell lines, clone formation assay and cell proliferation assay; 3. Bioinformatics analysis combined with luciferase reporter assay have demonstrated AML1-ET09a was a target gene of miR-9-1; fluorescence quantitative PCR was used to detect the expression of miR-9-1 in cell lines and clinical samples.Results:1. High-throughput sequencing data of exon showed that the average number of somatic mutations was 18.6 in each initially treated or recurrent sample (n=23). In the mutant gene reproducible statistical analysis that was conducted to identify potential common genetic mutations, the highest mutation frequency was observed in c-KIT (17.4%,4/23), followed by NRAS (13%,3/23), FLT3, NOTCH1 and PHF6 (8.7%,2/23, respectively). By summarizing findings from Suzhou University, and our analytical data of clinical samples and professor Dong-Er Zhang from UCSD (U.S.A), the proportion of AML1-ET09a in AML1-ETO positive clinical samples was proved to be 100%(44/44),100% (10/10) and 95%(35/37), respectively, far higher than that of c-KIT mutation; 2. In t (8; 21) AML, the total expression of SRSF was obviously higher than that of remaining AML clinical samples with chromosomes abnormalities. A further study found that splicing factors, SRSF2, SRSF6 and SRSF11, were over-expressed in AML1-ETO positive AML; 3. A signaling pathway analysis was carried out on 145 proteins with significant differences using RIP and mass spectrometry of ETO9a and its flanking sequences. The results revealed that 82%(119/145) of the proteins were closely related to mRNA nuclear splicing and machining and assembly and apoptosis of splicing complex. AMLl-ETO-positive Kasumi-1 and SKNO-1 cell lines transfected by SRSF2 lentivirus demonstrated that SRSF2 was capable of leading generation of AML1-ETO9a alternative splicing variant. Meanwhile, clone formation assay and cell proliferation experiments proved that SRSF2 played a role in specifically regulating malignant proliferation of AMLl-ETO positive cells but exhibited no effect on cell lines without AML1-ETO fusion gene, such as HL-60; 4. Bioinformatics analysis, luciferase reporter vector and Western blot assays demonstrated that AML1, AML1-ETO and AML1-ET09a were target genes of miR-9-1 which was expressed at a significantly lower level in t (8; 21) AML in real-time quantitative PCR assay.Conclusion:1. AML1-ET09a is a more extensive "double-hit" in t (8; 21) AML; 2. Splicing factor SRSF2 promotes alternative splicing of AML1-ET09a, and specifically accelerates malignant proliferation of cells; 3. In terms of translational level, AML1-ET09a is a target gene of miR-9-1, but the expression level of miR-9-1 is significantly decreased in t (8; 21) AML.Objective:Invasive fungal disease (IFD) causes morbidity and mortality in patients with hematological malignancy. Reactivation of IFD after chemotherapy or hematopoietic stem cell transplantation (HSCT) is associated with poor prognosis. The present study aimed to investigate the efficacy of different strategies of secondary antifungal prophylaxis (SAP) for IFD and choose an appropriate SAP regimen.Materials and methods:Clinical data of patients with previous IFD who underwent chemotherapy or HSCT between Jan 2008 and Jun 2013 were retrospectively reviewed and followed up to 180 days post-chemotherapy or HSCT. The clinical characteristics and diagnosis were analyzed according to the diagnostic criteria for IFD. The efficacy of different strategies for SAP and risk factors influencing the failure of SAP were evaluated.Results:Of the 164 patients enrolled,121 patients received SAP regimen (73.78%), and IFD recurred in 40 patients:16.5%(20/121) in SAP group and 46.5%(20/43) in non-SAP group. In SAP group,58 received SAP agents which were proven effective for their previous IFD, while other 63 patients received other broad-spectrum antifungal agents. There was no significant difference in the recurrence rates between these two subgroups (13.8%(8/58) vs 19.0%(12/63), P = 0.437). The IFD recurrence rates were statistically significant between patients with allogeneic HSCT and chemotherapy or autologous HSCT (25% vs 8.2%, P= 0.013). Multivariate analysis indicated that allogeneic HSCT was the independent risk factor of IFD recurrence after SAP.Conclusions:Secondary antifungal prophylaxis is necessary to prevent IFD recurrence in patients with hematological malignancy, especially for patients in the setting of allogeneic HSCT. |
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