The prognostic value of the peripheral blood cell counts changes during induction chemotherapy in Chinese patients with adult acute myeloid leukemia.

ABSTRACT: To investigate the prognostic value of the circulating peripheral blood cell counts changes in acute myeloid leukemia (AML) at different time points during induction chemotherapy.We retrospectively analyzed the clinical and laboratory data of 237 newly diagnosed AML patients admitted to Fujian Medical University Union Hospital from January 2011 to December 2014.1. When primitive cells were first removed from the circulating peripheral blood, it was called peripheral blood blast clearance (PBBC). These patients were divided into two groups, according to PBBC. Statistical analysis showed that the day 5 of induction chemotherapy was a better cut-off for PBBC. PBBC≤5 days is defined as early-blast-clearance, while PBBC >6 days is delayed-blast-clearance. There was significant difference between the two groups on complete remission (CR) rate (P = .002), recurrence-free survival (RFS) (P = .026) and overall survival (OS) (P = .001). 2. Multivariate analysis suggested PBBC is an independent prognostic factor for CR, RFS, and OS in AML. Receiver operating characteristic(ROC) curve analysis showed the CR rate of patients with white blood cell count less than 1.25 × 109/L was significantly higher than that of patients with white blood cell count more than 1.25 × 10 9/L (P < .001) at day 5 of induction chemotherapy, but the RFS and OS was no significantly different (P > .05).The dynamics of peripheral blood blast in AML after initiation of induction chemotherapy, especially the time length to achieve PBBC, has important prognostic value for CR rate, RFS, and OS in AML patients. It is a simple and feasible method to evaluate the efficacy of AML.

Lentivirus-mediated RNA interference targeting FAMLF-1 inhibits cell growth and enhances cell differentiation of acute myeloid leukemia partially differentiated cells via inhibition of AKT and c-MYC.

Genetic heterogeneity is the basis of clinical heterogeneity among different subtypes of AML. We have successfully cloned a gene related to AML termed FAMLF from a FAB-M2 patient's sample of a second largest AML pedigree. Then we revealed at least three splice variants, named as FAMLF-1, FAMLF-2 and FAMLF-3, and found miR181a1/b1 in the second intron of FAMLF gene family. Higher expression of FAMLF-1 was related to a higher complete remission (CR) rate, but shorter relapse free survival (RFS) in AML. We further found that the FAMLF-1 single nucleotide polymorphism (SNP) haplotype and its expression were positively correlated to clinical parameters of acute myeloid leukemia partially differentiated (FAB-M2) patients, but not FAB non-M2 patients or Acute Monocytic Leukemia (FAB-M5) patients. GTAGG SNP haplotype of FAMLF gene might increase FAB-M2 susceptibility in Han population and act as a useful candidate biomarker for FAB-M2 screening. We also demonstrated that FAMLF-1 gene silencing in FAB-M2 cells could lead to proliferation inhibition, cell cycle G0/G1 phase arrest, and differentiation promotion independent of its intronic miR-181a1, which might be related to Akt/c-Myc pathway. These findings reveal a role of FAMLF-1 as a potential pathogenic gene for FAB-M2.

DHX15 is associated with poor prognosis in acute myeloid leukemia (AML) and regulates cell apoptosis via the NF-kB signaling pathway.

The role of DHX15, a newly identified DEAH-box RNA helicase, in leukemogenesis remains elusive. Here, we identified a recurrent mutation in DHX15 (NM_001358:c.664C>G: p.(R222G)) in one familial AML patient and 4/240 sporadic AML patients. Additionally, DHX15 was commonly overexpressed in AML patients and associated with poor overall survival (OS) (P=0.019) and relapse-free survival (RFS) (P=0.032). In addition, we found a distinct expression pattern of DHX15. DHX15 was highly expressed in hematopoietic stem cells and leukemia cells but was lowly expressed in mature blood cells. DHX15 was down-regulated when AML patients achieved disease remission or when leukemia cell lines were induced to differentiate. DHX15 silencing greatly inhibited leukemia cell proliferation and induced cell apoptosis and G1-phase arrest. In contrast, the restoration of DHX15 expression rescued cell viability and reduced cell apoptosis. In addition, we found that DHX15 was down-regulated when cell apoptosis was induced by ATO (arsenic trioxide); overexpression of DHX15 caused dramatic resistance to ATO-induced cell apoptosis, suggesting an important role for DHX15 in cell apoptosis. We further explored the mechanism of DHX15 in apoptosis and found that overexpression of DHX15 activated NF-kB transcription. Knockdown of DHX15 inhibited the nuclear translocation and activation of the NF-kB subunit P65 in leukemia cells. Several downstream targets of the NF-kB pathway were also down-regulated, and apoptosis-associated genes CASP3 and PARP were activated. In conclusion, this study represents the first demonstration that DHX15 plays an important role in leukemogenesis via the NF-kB signaling pathway and may serve as an independent prognostic marker for AML.

[Levels and Significance of Serum Interleukin-35 and the New Regulatory T Cells -iTR35 Cells in Myelodysplastic Syndrome Patients].

OBJECTIVE: To investigate the significance of serum interleukin-35 level and the new regulatory T cells -iTR35 cells in patients with myelodysplastic syndrome (MDS). METHODS: Twenty three cases of newly diagnosed MDS were enrolled in this study from January 2014 to January 2016 in Department of Hematology of The First Hospital of Quanzhou in Fujian Province. According to MDS International Prognostic Scoring System (IPSS), the 23 patients were divided into 4 groups: high-risk (n=4), intermediate risk-2 (n=10), intermediate risk-1 (n=5) and low-risk group(n=4). Twenty healthy people of routine physical examination were used as control during the same period. Enzyme Linked Immunosorbent assay(ELISA) and flow cytometry(FCM) were used to detect the expression level of serum IL-35, the proportion of iTR35 cells, and the expression levels of associated molecules such as IL-12p35,IL-27EBl3 respectively. RESULTS: The proportion of CD4+ CD25+ Foxp3+ Treg cells in peripheral blood of MDS patients was significantly higher than that in controls (P<0.01). The proportion of iTR35 cells was also higher than that in controls(P<0.01). However, the proportion of CD4+ CD25- Foxp3+ T cells was not significantly different between 2 groups (P>0.05). The level of serum IL-35 in MDS patients was significantly higher than that in control group (P<0.05). The expression levels of IL-12p35 and IL-27EBl3 in the Treg cells were also significantly upregulated than those in control group(P<0.05), the expression levels of IL-35, IL-12p35 and IL-27EBl3 in MDS group positively correlated with the proportion of iTR35 cells(r=0.92, 0.99 and 0.52, P<0.05, respectively). IL-35 level and the proportion of iTR35 cells in 4 groups of MDS showed significantly difference in general term, no significant difference was found in IL-35 level between the high-risk group and intermediate risk-2 group (P>0.05), but the IL-35 levels in both groups were significantly higher than those in intermediate risk-1 group and the low-risk group (P<0.05), and the level in the intermediate risk-2 group was significantly higher than that in the intermediate risk-1 group and the low-risk group (P<0.05), while there was not different between the intermediate risk-1group and the low risk group (P>0.05). The proportion of iTR35 cells was not significantly different between the high-risk group and the intermediate risk-2 group. The proportions of iTR35 cells in the high-risk group and the intermediate risk-2 group were higher than those in the intermediate risk-1 group and the low-risk group respectively (P<0.05), but there was no differentce in population of iTR35 between the intermediate risk-1 group and the low-risk group (P>0.05). CONCLUSION: The imbalance between IL-35 level and iTR35 cells propertion may play an important role in the development of MDS, which possibly to provides a new theoretical basis for the study of MDS immune targeting therapy.

[Expression Analysis and Epigenetics of MicroRNA let-7b in Acute Lymphoblastic Leukemia].

OBJECTIVE: To study the expression and its mechamisms of microRNA let-7b in adult acute lymphoblastic leukemia (ALL), so as to provide the basis for searching a new targeted therapy. METHODS: Firstly, methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of CpG islands in microRNA let-7b promoter of bone marrow mononuclear cells in the patients with ALL and patients with non-hematologic malignancies as control, the real-time fluorescence quantitative polymerase chain reaction (qPCR) was used to detect the expression levels of microRNA let-7b in this 2 groups; and then 5-aza-2'-deoxycytidine (5-Aza-dC, DAC) was used to treat ALL cell line MOLT-4; after drug treatment, MSP was used to analyze the methylation status of the CpG islands in microRNA let-7b promoter; the qPCR was used to detect the expression levels of microRNA let-7b, and further explore the regulatory mechanism of microRNA let-7b expression. RESULTS: Hypermethylation of CpG islands in microRNA let-7b promoter in ALL patients was significantly higher than that in patients with non-hematologic malignancies, and the relative expression level of microRNA let-7b was significantly reduced in ALL patients; 5-aza-dC could significantly inhibit the growth of MOLT-4 cells and arrest the cells in G1 phase, thus biosynthesis of RNA and protein was suppressed, and the apoptosis was promoted, meanwhile, 5-Aza-dC could increase the expression of microRNA let-7b. CONCLUSION: In the patients with ALL, the expression of microRNA let-7b is regulated by methylation of CpG islands in the region of genomic promoter. The microRNA let-7b may act as a tumor suppressor, whose low expression is involved in ALL development, indicating the microRNA let-7b may become a new therapeutic target for ALL.

Positional cloning and next-generation sequencing identified a TGM6 mutation in a large Chinese pedigree with acute myeloid leukaemia.

An inherited predisposition to acute myeloid leukaemia (AML) is exceedingly rare, but the investigation of these families will aid in the delineation of the underlying mechanisms of the more common, sporadic cases. Three AML predisposition genes, RUNX1, CEBPA and GATA2, have been recognised, but the culprit genes in the majority of AML pedigrees remain obscure. We applied a combined strategy of linkage analysis and next-generation sequencing (NGS) technology in an autosomal-dominant AML Chinese family with 11 cases in four generations. A genome-wide linkage scan using a 500K SNP genotyping array was conducted to identify a previously unreported candidate region on 20p13 with a maximum multipoint heterogeneity LOD (HLOD) score of 3.56 (P=0.00005). Targeted NGS within this region and whole-exome sequencing (WES) revealed a missense mutation in TGM6 (RefSeq, NM_198994.2:c.1550T>G, p.(L517W)), which cosegregated with the phenotype in this family, and was absent in 530 healthy controls. The mutated amino acid was located in a highly conserved position, which may be deleterious and affect the activation of TGM6. Our results strongly support the candidacy of TGM6 as a novel familial AML-associated gene.

[Proliferation promotion and apoptotic inhibition effects of ribosomal protein RPL36A small interference RNA on U937 cells].

This study was purposed to investigate the expression of RPL36A (ribosomal protein 36a) in the newly diagnosed acute myeloid leukemia (AML) cells and its mechanism at the molecular level. The RPL36A mRNA expression in the newly diagnosed AML cells, U937 cells and normal MNCs was determined by RT-PCR. Small interfering RNA (siRNA) targeting to RPL36A was transfected into U937 cells by Lipofectamine 2000 system. Proliferation, cell cycle, apoptosis of U937 were observed through MTT assay, flow cytometry, acridine orange/ethidium bromide (AO/EB) double staining, TUNEL and Annexin V/FITC respectively. RPL36A mRNA and protein expression levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis respectively. The results showed that RPL36A expression in the newly diagnosed AML cells and U937 cells was significantly upregulated. The average OD value of U937 cells transfected with RPL36A siRNA was significantly lower as compared with 3 control groups. The cell percentage in G2-and S-phase increased, which indicated the inhibition effect of RPL36A siRNA on cell proliferation. Remarkable cell apoptosis in U937 cells treated with RPL36A siRNA was observed by AO/EB, TUNEL analysis and Annexin V/FITC assay; RPL36A mRNA and protein expression level of U937 cells treated with siRNA were significantly declined in a time-dependent manner (r=0.9813 and 0.9537). It is concluded that the RPL36A expression in the AML cells is significantly enhanced and the RPL36A gene may be involved in regulation of cell cycle and cell apoptosis of AML, which promotes proliferation of AML cells and inhibits apoptosis of cells.

[Full-length cDNA cloning and biological function analysis of a novel gene FAMLF related to familial acute myelogenous leukemia].

OBJECTIVE: To clone the full-length cDNA of a novel gene related to familial acute myelogenous leukemia (AML) and to demonstrate its molecular mechanisms on the gene level. METHODS: Bone marrow specimen was obtained from a patient of familial AML, male, aged 11, and peripheral blood samples were obtained from 23 AML patients outside this family, 9 normal persons in this family, and 23 normal persons outside this family. Based on the EST sequence zywb87 (GenBank accession number: CV973101) from a subtractive cDNA library of differential expressed genes constructed in familial AML, SMART-rapid amplification of cDNA ends (SMART-RACE) was applied to clone the full-length cDNA of the novel gene, and bioinformatics was used to predict its biological function, the expression of the novel gene in AML was detected by One-Step RT-PCR. RESULTS: A full-length cDNA of 2313 bp was obtained from the bone marrow specimen of the familial AML patient with complete open reading frame (ORF) of 249 bp. Localized on 1q31.3 of human chromosome, it coded a 82-amino acid polypeptide with signal peptide, leucine-rich repeat (LRR_SD22), and intrinsic disorder functional domain. BLAST analysis confirmed this gene as a novel gene designated with the accession number: (nucleotide) EF413001 and (protein) ABN58747 by GenBank and was named as Homo sapiens familial acute myelogenous leukemia related factor (FAMLF). The FAMLF expression level of the AML patients outside this family was (2.61 +/- 0.66), significantly higher than that of the normal persons outside this family (0.97 +/- 0.51, P < 0.01). CONCLUSION: A full-length cDNA of the novel gene FAMLF related to familial AML has been obtained. The FAMLF gene is expressed highly in AML and may present biological function on the progress of AML.