Background The receptor tyrosine kinase FLT3 with internal tandem duplications within the juxtamembrane domain ( FLT3 -ITD) is a poor prognostic factor; however, the prognostic significance of missense mutation in the tyrosine kinase domain ( FLT3 -TKD) is controversial. Furthermore, the accompanying mutations and fusion genes with FLT3 mutations are unclear in acute myeloid leukemia (AML). Methods We investigated FLT3 mutations and their correlation with other gene mutations and gene fusions through two RNA-seq based next-generation sequencing (NGS) method and prognostic impact in 207 de novo AML patients. Results FLT3 -ITD mutations were positive in 58 patients (28%), and FLT3 -TKD mutations were positive in 20 patients (9.7%). FLT 3-ITD was associated with a higher white blood cell count (WBC, mean 72.9 × 10 9 /L vs. 24.2 × 10 9 /L, P = 0.000), higher bone marrow blasts (mean 65.9% vs. 56.0%, P = 0.024), and NK-AML (normal karyotype) (64.8% vs. 48.4%, P = 0.043). NPM1 and DNMT3A mutations were enriched in FLT3 -ITD (53.5% vs. 15.3%, P = 0.000; 34.6% vs. 13%, P = 0.003). However, the mutations of CEBPA were excluded in FLT3 -AML (3.8% vs. 0% vs. 19.8%, P = 0.005). Mutations of Ras and TP53 were unlikely associated with FLT3 -ITD (1.9% vs. 20.6%, P = 0.006; 0% vs. 6.1%, P = 0.04). The common fusion genes (> 10%) in FLT3 -ITD had MLL- rearrangement and NUP98 -rearrangement, while the common fusion genes in FLT3 -TKD had AML1-ETO and MLL -rearrangement. Two novel fusion genes PRDM16-SKI and EFAN2-ZNF238 were identified in FLT3-ITD patients. Gene fusions and NPM1 mutation were mutually excluded in FLT3 -ITD and FLT3 -TKD patients. Their patterns of mutual exclusivity and cooperation among mutated genes suggest that additional driver genetic alterations are required and reveal two evolutionary patterns of FLT3 pathogenesis. Patients with FLT3 -ITD had a lower CR (complete remission) rate, lower 3-year OS (overall survival), DFS (disease-free survival), and EFS (event-free survival) compared to FLT3 wt AML. NK-AML with FLT3 -ITD had a lower 3-year OS, DFS, and EFS than those without, while FLT3 -TKD did not influence the survival in whole cohort and NK-AML. Besides, we found that FLT3 -ITD/ TET2 bimutation defined a poor prognostic subgroup. Conclusions Our study offers deep insights into the molecular pathogenesis and biology of AML with FLT3 -ITD and FLT3 -TKD by providing the profiles of concurrent molecular alterations and the clinical impact of FLT3 -ITD and FLT3 -TKD on AML patients.

The t(8;21) fusion product, AML1/ETO, and hypoxia-inducible factor 1α (HIF1α) form a feed-forward transcription loop that cooperatively transactivates the DNA methyltransferase 3a gene promoter that leads to DNA hypermethylation and drives leukemia cell growth. Suppression of the RNA N6-methyladenosine (m6A)-reader enzyme YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) specifically compromises cancer stem cells in acute myeloid leukemia (AML) but promotes hematopoietic stem cell expansion without derailing normal hematopoiesis. However, the relevance of expression between AML1/ETO-HIF1α loop and YTHDF2, and its functional relationship with t(8;21) AML have not been documented. Here, we show that YTHDF2 is highly expressed in t(8;21) AML patients and associated with a higher risk of relapse and inferior relapse-free survival. Knockdown of YTHDF2 in leukemia cells causes an impaired cell proliferation rate in vitro and in mice. Mechanistically, HIF1α is able to bind to the hypoxia-response elements of the 5′-untranslated region of the YTHDF2 gene and promotes the transactivity of the YTHDF2 promoter. Knockdown and overexpression of either AML1/ETO or HIF1α resulted in decreased and increased YTHDF2 protein and mRNA expression in t(8;21) AML cells. In particular, knockdown of YTHDF2 resulted in increased global mRNA m6A levels in t(8;21) AML cells, accompanied by increased TNF receptor superfamily member 1b (TNFRSF1b) mRNA and protein expression levels. Last, we demonstrated that the m6A methylation and expression levels of the TNFRSF1b gene were both negatively correlated with HIF1α expression levels. In conclusion, YTHDF2 is a downstream target of the AML1/ETO-HIF1α loop and promotes cell proliferation probably by modulating the global m6A methylation in t(8;21) AML.

Single-cell RNA sequencing (scRNA-seq) of bone marrow or peripheral blood samples from patients with acute myeloid leukemia (AML) enables the characterization of heterogeneous malignant cells. A total of 87 cells from two patients with t(8;21) AML were analyzed using scRNA-seq. Clustering methods were used to separate leukemia cells into different sub-populations, and the expression patterns of specific marker genes were used to annotate these populations. Among the 31 differentially expressed genes in the cells of a patient who relapsed after hematopoietic stem cell transplantation, 13 genes were identified to be associated with leukemia. Furthermore, three genes, namely AT-rich interaction domain 2, lysine methyltransferase 2A and synaptotagmin binding cytoplasmic RNA interacting protein were validated as possible prognostic biomarkers using two bulk expression datasets. Taking advantage of scRNA-seq, the results of the present study may provide clinicians with several possible biomarkers to predict the prognostic outcomes of t(8;21) AML.

BACKGROUND Haplo-identical hematopoietic stem cell transplantation (HSCT) has provided potential donors for patients lacking available HLA-matched donors. ABO blood type compatibility has been reported to be associated with HSCT outcomes. However, few studies have investigated the role of ABO compatibility in haplo-identical HSCT of AML patients. MATERIAL AND METHODS We retrospectively analyzed 42 adult acute myeloid leukemia (AML) patients who received unmanipulated haplo-identical peripheral blood HSCT at the Chinese PLA General Hospital between Jan 2013 and Dec 2017. We analyzed the role of ABO compatibility in engraftment, transfusion requirements, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viremia, acute graft-versus-host disease (GVHD), overall survival (OS), transplantation-related mortality (TRM), relapse, chronic GVHD, and post-transplant lymphoproliferative disorder (PTLD). RESULTS There were no significant differences between the ABO-matched group and the ABO-mismatched group in terms of engraftment, transfusion requirements, CMV and EBV viremia, OS, TRM, relapse, PTLD, and chronic GVHD. Univariate analysis revealed ABO incompatibility is not an independent risk factor of engraftment, transfusion requirements, CMV and EBV viremia, OS, TRM, relapse, PTLD, and chronic GVHD. We found a significantly higher cumulative incidence of aGVHD in the matched group compared with the mismatched group (80.95% vs. 42.86%, p=0.020). In multivariate analysis, ABO mismatch was associated with decreased risk of acute GVHD within 100 days after transplant (hazard ratio 0.492, 95% confidence interval 0.2123-1.14). However, the difference was not statistically significant (p=0.099). CONCLUSIONS This study demonstrated ABO incompatibility is not an independent risk factor of outcomes for AML patients who received unmanipulated haplo-identical peripheral blood HSCT. ABO compatibility might have limited value in haplo-identical donor selection.

Background Post-transplant lymphoproliferative disorder (PTLD) is a rare complication following solid organ transplantation and allogeneic hematopoietic stem cell transplantation (Allo-HSCT), which gives rise to high mortality rates. Material/Methods This was a single-center retrospective analysis based on 27 patients who were diagnosed with PTLD following Allo-HSCT between January 1, 2007 and June 2018 at the Chinese PLA General Hospital. The purpose of this analysis was to investigate responses and prognostic factors of rituximab-based treatment. Results Twenty-seven patients were treated with rituximab. Among them, 20 of 27 patients (74.07%) had a complete response, 2 of 27 patients (7.41%) had a partial response, 5 of 27 patients (18.52%) had no response, and 22 of 27 patients (81.48%) cleared Epstein-Barr virus (EBV) copies. There were no obvious side effects. The 1-year overall survival (OS) estimate was 46.8% (95% CI, 23.1–65.5%). Univariate analysis revealed that lower OS was correlated with Eastern Cooperative Oncology Group (ECOG) score standard (3–4), Epstein-Barr virus (EBV) viral load (≥106 copies/mL), bacteria or fungal infection, and EBV reactivation were positive after treatment with 1 or 2 doses of rituximab (P<0.05). Multivariate analysis showed that each of the following were independently associated with lower OS (P<0.05): female, ECOG score standard (3–4), and EBV reactivation were positive after treatment with 1 or 2 doses of rituximab. Conclusions Our results demonstrated that rituximab-based treatment was a safe and effective strategy for patients who were diagnosed with PTLD following Allo-HSCT. The identified prognostic factors may help to detect which PTLD patients are at a higher risk of mortality.

OBJECTIVE:To explore the frequency and spectrum of thalassemia gene mutations of the population in Sanya area of Hainan province in China.METHODS:The type and frequency of gene mutation in 1060 patients with suspected thalassemia were analyzed by Gap-PCR and reverse dot blot (RDB).RESULTS:The detection on mutation of thalassemia gene were found in 539 suspected thalassemia patients, the total detected rate was 50.85% (539/1060), out of them 330 (31.13%) were diagnosed with α-thalassemia, 162 (15.28%) with β-thalassemia, and 47 (4.43%) as carriers of both α and β-thalassemia. In α-thalassemia patients, genotype were as follows in proper order--SEA/αα (9.25%)、-α 4.2/αα (5.94%)，HbH (5.56%)，-α 3.7/αα (5.00%)，-α 3.7/-α 4.2 (2.36%)，-α 4.2/-α 4.2 (1.70%), and -α3.7/-α3.7(1.32%). In β-thalassemia patients, there were 9 gene mutations: CD41-42 (9.8%), CD17 (1.32%), 654 (1.23%), CD71-72 (1.23%), IVS-II-654 (1.04%), -28 (0.37%), CD43 (0.19%), -29 (0.18%) and βE (0.09%). In the α and β composite thalassemia there were 12 genotypes. The -α3.7/αα was the most common genotype co-existed with β-thalassemia (1.70%), followed by the -α 4.2 /αα genotype (0.94%).CONCLUSION:The data of this study provide the frequency and the spectrum of thalassemia gene mutations in the sanya area of Hainan province, which can contribute to set up the strategies for the prevention and control of thalassemia in this area.

This study aimed to develop a novel multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) assay to accurately and effectively detect 10 common gene rearrangements in adult acute lymphoblastic leukemia (ALL) and to examine the clinicopathologic characteristics and other genetic aberrations of patients with ALL expressing different fusion genes. Our RT-nPCR assay had a positive detection rate of 35.15% (90/256) for the 10 fusion genes. BCR-ABL1, FUS-ERG, MLL-AF4, ETV6-RUNX1, E2A-PBX1, dupMLL, MLL-AF10, MLL-ENL, SET-NUP214 and SIL-TAL1 were detected in 36 (14.06%), 14 (5.47%), 14 (5.47%), four (1.56%), four (1.56%), five (1.95%), four (1.56%), two (0.78%), two (0.78%) and five patients (1.95%), respectively. The RT-nPCR results were further confirmed by split-out PCR, and cytogenetic and fluorescence in situ hybridization (FISH) analysis revealed corresponding translocations and fusions in 63 and 74 cases, respectively. JAK2 and IKZF1 mutations were commonly detected in patients with BCR-ABL1 ALL, and HOX overexpression was highly correlated with MLL fusions and SET-NUP214. This study demonstrates that RT-nPCR is an effective method for identifying 10 gene rearrangements in adult ALL, and it could potentially be developed for diagnostic use and prognostic studies of ALL.

This study was aimed to evaluate the prevalence of FT3-ITD mutation in patients with acute myeloid leukemia (AML) and its significance for prognosis. The bone marrow of 216 patients with AML were detected by using PCR and R-banding methods. On basis of patients' clinical data the clinical feature and prognosis were analyzed. The results showed that in 216 AML patients incidence of FLT3-ITD mutation was 20.83%. FLT3-ITD mutation was present in 24% patients with normal karyotype while in 12.5% patients with abnormal karyotype. The FLT3-ITD positive patients had more white blood cells in peripheral blood and leukemia cells in bone marrow (P < 0.01). The free-disease survival and overall survival time were shorter in FLT3-ITD positive cases than that in FLT3-ITD negative cases (P < 0.05). It is concluded that FLT3-ITD mutation positive patients have more white blood cells in peripheral blood and leukemia cells in bone marrow. The FLT3-ITD mutation may be a danger factor in the AML patients with normal karyotype. It may be helpful for evaluating prognosis and guiding individual treatment of AML.

The study was purposed to explore the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) combined with Imatinib for treatment of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph(+)ALL) patients. From 2007 to 2008, 3 patients with Ph(+)ALL were treated with allogeneic hematopoietic stem cell transplantation and Imatinib, and the follow-up ended at Oct 21(st) 2009. 1 patient received HSCT from matched sibling donor and 2 patients from haploidentical related donors. All 3 patients achieved complete remission before transplantation and were treated with Imatinib for distinct time at different periods before and/or after transplantation. The level of bcr/abl mRNA was monitored using real-time PCR. The results showed that all 3 patients achieved stable engraftments without severe transplantation related complications. The level of bcr/abl mRNA declined and achieved zero level finally. In conclusion, the allo-HSCT combined with Imatinib is an effective therapy regimen for Ph(+)ALL patients.