Purpose: APL patients have recurrent alterations in FLT3, WT1, NRAS and KRAS. Gene mutations have a strong potential for involvement in pathogenesis and may have potential effects on the clinical manifestations. Gene mutations may even be associated with early death (ED) in APL patients. However, there is little published information on mutations in APL patients and whether they are attributed to early death.

Methods: In this study, we retrospectively analyzed the clinical data and gene mutations of 134 de novo APL patients. We detected the gene mutations by next-generation sequencing (NGS) to investigate the genetic predictors of early death in APL patients. According to the number of gene mutations per patient, the 134 APL patients were divided into three groups. All patients received arsenic trioxide (ATO) alone as induction therapy. The clinical data and gene mutations were compared and analyzed.

Results: A total of 134 APL patients were involved in the study. The clinical data of sex, WBC, PT, and DD, UA, and LDH level were significantly different between the three groups (P = 0.000, P = 0.000, P = 0.009, P = 0.020, P = 0.030, P = 0.001 and P = 0.014, respectively). Meanwhile, among them, the Sanz risk stratification and early death rate were significantly different (P = 0.001). The early death rate was 10.4%, and the median time to early death was 6.6 days (range 2-15 days). For the next-generation sequencing, a mean of 1.28 ± 1.06 mutations per patient was detected (range: 0-5). The univariate and the multivariate regression analysis showed that age > 50[HR = 1.666, CI (1.027-2.702), P = 0.039], high WBC count [HR = 4.702, CI (1.026-21.543), P = 0.046] and low ALB levels [HR = 4.547, CI (1.088-18.995), P = 0.038] were independent risk factors for early death in APL patients. Furthermore, Kaplan-Meier survival analysis, univariate analysis, and the multivariate regression analysis showed that patients with multiple gene mutations [HR = 2.258, CI (1.115-4.571), P = 0.024], KRAS [HR = 5.136, CI (1.356-19.455), P = 0.016] and/or GATA2 [HR = 4.070, CI (1.287-12.877), P = 0.017] have a significantly higher early death rate.

Conclusion: The results of this investigation show that both molecular markers and clinical variables should be used as potential predictors for early death in APL patients. Our results suggested that age > 50, high WBC count, low ALB levels, and the presence of multiple gene mutations, KRAS and/or GATA2 at the time of diagnosis were independent risk factors for early death in APL patients. For these patients, clinicians should be more cautious during the course of induction treatment.

Keywords: Acute promyelocytic leukemia; Early death; GATA2; Gene mutations; KRAS; Next-generation sequencing.

WHIM syndrome is a condition in which affected persons have chronic peripheral neutropenia, lymphopenia, abnormal susceptibility to human papilloma virus infection, and myelokathexis. Myelokathexis refers to the retention of mature neutrophils in the bone marrow (BM), which accounts for degenerative changes and hypersegmentation. Most patients present heterozygous autosomal dominant mutations of the gene encoding CXCR4. Consequently, aberrant CXCL12/CXCR4 signaling impairs the receptor downregulation causing hyperactivation (gain-of-function) that affects BM homing for myelopoiesis and lymphopoiesis and the release of neutrophils in the bloodstream. We report the case of a 26-year-old female with severe foot and hand cutaneous warts since childhood, recalcitrant genital condylomatas, bacterial infections, and intraepithelial cervical neoplasia. Laboratory tests revealed severe B lymphopenia and HPV high and low risk types. HIV testing was negative. Not only CXCR4 but also GATA2, NEMO, and CD40L gene mutations were excluded. BM smears revealed, in the presence of a normal cellularity, hyperplasia of myeloid cells (MPO positive) and karyorrhexis, especially in neutrophils and eosinophils. Of note, neutrophils with altered lobation of nuclei connected by long thin chromatin filaments were observed. Our patient presented a clinical and histological picture reminiscent of WHIM in the presence of normal peripheral neutrophil counts and wild-type CXCR4 gene. Although the BM did not reveal a classical pattern of myelokathexis, the observation of consistent signs of neutrophil dysplasia has fuelled the hypothesis of a novel WHIM variant or a novel immunodeficiency. We speculate that abnormalities that affect CXCR4/CXCL12 pair, including GRK levels or activity, might be responsible for this WHIM-like disorder.

Introduction: While the acquisition of mutations in the ABL1 kinase domain (KD) has been identified as a common mechanism behind tyrosine kinase inhibitor (TKI) resistance, recent genetic studies have revealed that patients with TKI resistance or intolerance frequently harbor one or more genetic alterations implicated in myeloid malignancies. This suggests that additional mutations other than ABL1 KD mutations might contribute to disease progression.

Methods: We performed targeted-capture sequencing of 127 known and putative cancer-related genes of 63 patients with CML using next-generation sequencing (NGS), including 42 patients with TKI resistance and 21 with TKI intolerance.

Results: The differences in the number of mutations between groups had no statistical significance. This could be explained in part by not all of the patients having achieved major molecular remission in the early period as expected. Overall, 66 mutations were identified in 96.8% of the patients, most frequently in the KTM2C (31.82%), ABL1 (31.82%), FAT1 (25.76%), and ASXL1 (22.73%) genes. CUX1, KIT, and GATA2 were associated with TKI intolerance, and two of them (CUX1, GATA2) are transcription factors in which mutations were identified in 82.61% of patients with TKI intolerance. ASXL1 mutations were found more frequently in patients with ABL1 KD mutations (38.1% vs 15.21%, P=0.041). Although the number of mutations was low, pairwise interaction between mutated genes showed that ABL1 KD mutations cooccurred with SH2B3 mutations (P<0.05). In Kaplan-Meier analyses, only TET2 mutations were associated with shorter progression-free survival (P=0.026).

Conclusion: Our data suggested that the CUX1, KIT, and GATA2 genes may play important roles in TKI intolerance. ASXL1 and TET2 mutations may be associated with poor patient prognosis. NGS helps improving the clinical risk stratification, which enables the identification of patients with TKI resistance or intolerance in the era of TKI therapy.

Keywords: chronic myeloid leukemia; intolerance; mutations; resistance; tyrosine kinase inhibitor.

The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.

Colorectal cancer (CRC) is one of the most common cancers. Almost 1/3 of CRC are rectal cancer, and 95% of rectal cancers are rectal adenocarcinoma (READ). Increasing evidences indicated that long noncoding RNAs (lncRNAs) have important role in the genesis and development of cancers. The purpose of our present study was to identify the differential expression lncRNAs which potentially related with immune cells infiltration and establish a risk assessment model to predict the clinical outcome for READ patients. We obtained three immune-related differential expression lncRNAs (IR-DELs) (C17orf77, GATA2-AS1, and TPT1-AS1) by differential expression analysis following correlation analysis and Cox regression analysis. A risk assessment model were constructed by integrating these analysis results. We then plotted the 1-, 3-, and 5-year ROC curves depending on our risk assessment model, which suggested that all AUC values were over 0.7. In addition, we found that the risk assessment model was correlated with several immune cells and factors. This study suggested that those three signatures (C17orf77, GATA2-AS1, and TPT1-AS1) screened by pairing IR-DELs could be prognosis markers for READ patients and might benefit them from antitumor immunotherapy.

Keywords: LncRNAs; READ; immune; overall survival; risk model.

Biallelic mutations of the CEBPA gene (CEBPAbi) define a distinct entity associated with favorable prognosis, however the role of monoallelic mutations (CEBPAsm) is poorly understood. We retrospectively analyzed 4708 adult AML patients recruited into Study Alliance Leukemia trials to investigate the prognostic impact of CEBPAsm. CEBPA mutations were identified in 240 patients (5.1%), 131 CEBPAbi and 109 CEBPAsm (60 affecting the amino-terminal transactivation domains (CEBPAsmTAD) and 49 the carboxy-terminal DNA-binding or basic leucine zipper region (CEBPAsmbZIP)). Interestingly, CEBPAbi and CEBPAsmbZIP patients shared several clinical factors, i.e. were significantly younger (median 46 years and 50 years) and had higher WBC counts at diagnosis (median 23.7 and 35.7 109/l) compared to CEBPAsmTAD patients (median age 63 yrs., median WBC 13.1 109/l; p<.001). Co-mutations were also similar in both groups, e.g. GATA2 mutations (35.1% CEBPAbi; 36.7% CEBPAsmbZIP vs. 6.7% CEBPAsmTAD; p<.001) or NPM1 mutations (3.1% CEBPAbi; 8.2% CEBPAsmbZIP vs. 38.3% CEBPAsmTAD; p<.001). CEBPAbi and CEBPAsmbZIP, but not CEBPAsmTAD were associated with significantly improved overall (median OS: 103 and 63 vs. 13 months) and event-free survival (median EFS: 20.7 and 17.1 vs. 5.7 months), in univariate and multivariable analyses. More detailed analysis revealed that the clinical and molecular features as well as the favorable survival were confined to patients showing in-frame mutations in bZIP (CEBPAbZIP-inf). When grouping patients into CEBPAbZIP-inf and CEBPAother (including CEBPAsmTAD and other non-CEBPAbZIP-inf patients), only CEBPAbZIP-inf patients showed superior CR rates and the longest median OS and EFS, arguing for a previously undefined prognostic role of this type of mutations.

Purpose: The clinical and inflammatory associations of mast cells (MCs) and basophils in chronic obstructive pulmonary disease (COPD) are poorly understood. We previously developed and validated a qPCR-based MC/basophil gene signature in asthma to measure these cells in sputum samples. Here, we measured this gene signature in a COPD and control population to explore the relationship of sputum MCs/basophils to inflammatory and COPD clinical characteristics.

Patients and methods: MC/basophil signature genes (TPSAB1/TPSB2, CPA3, ENO2, GATA2, KIT, GPR56, HDC, SOCS2) were measured by qPCR in sputum from a COPD (n=96) and a non-respiratory control (n=17) population. Comparative analyses of gene expression between the COPD and the control population, and between eosinophilic COPD and non-eosinophilic COPD were tested. Logistic regression analysis and Spearman correlation were used to determine relationships of sputum MC/basophil genes to inflammatory (sputum eosinophil proportions, blood eosinophils) and clinical (age, body mass index, quality of life, lung function, past year exacerbations) characteristics of COPD.

Results: MC/basophil genes were increased in COPD versus control participants (CPA3, KIT, GATA2, HDC) and between eosinophilic-COPD and non-eosinophilic COPD (TPSB2, CPA3, HDC, SOCS2). We found all MC/basophil genes were positively intercorrelated. In COPD, MC/basophil genes were associated with eosinophilic airway inflammation (GATA2, TPSB2, CPA3, GPR56, HDC, SOCS2), blood eosinophilia (all genes) and decreased lung function (KIT, GATA2, GPR56, HDC).

Conclusion: We demonstrate associations of MCs and basophils with eosinophilic inflammation and lower lung function in COPD. These findings are consistent with prior results in asthma and may represent a new tool for endotyping eosinophilic-COPD.

Keywords: COPD; basophils; gene expression; inflammation; mast cells.

Hematopoietic stem and progenitor cells (HSPCs) are attractive targets in regenerative medicine, although the differences in their homeostatic maintenance between sexes along time are still under debate. We accurately monitored hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs) frequencies by flow cytometry, by performing serial peripheral blood extractions from male and female B6SJL wild-type mice and found no significant differences. Only modest differences were found in the gene expression profile of Slamf1 and Gata2. Our findings suggest that both sexes could be used indistinctly to perform descriptive studies in the murine hematopoietic system, especially for flow cytometry studies in peripheral blood. This would allow diminishing the number of animals needed for the experimental procedures. In addition, the use of serial extractions in the same animals drastically decreases the number of animals needed.

In Xenopus laevis, bone morphogenetic proteins (Bmps) induce expression of the transcription factor Gata2 during gastrulation, and Gata2 is required in both ectodermal and mesodermal cells to enable mesoderm to commit to a hematopoietic fate. Here, we identify tril as a Gata2 target gene that is required in both ectoderm and mesoderm for primitive hematopoiesis to occur. Tril is a transmembrane protein that functions as a co-receptor for Toll-like receptors to mediate innate immune responses in the adult brain, but developmental roles for this molecule have not been identified. We show that Tril function is required both upstream and downstream of Bmp receptor-mediated Smad1 phosphorylation for induction of Bmp target genes. Mechanistically, Tril triggers degradation of the Bmp inhibitor Smad7. Tril-dependent downregulation of Smad7 relieves repression of endogenous Bmp signaling during gastrulation and this enables mesodermal progenitors to commit to a blood fate. Thus, Tril is a novel component of a Bmp-Gata2 positive-feedback loop that plays an essential role in hematopoietic specification.

Human embryonic stem cells (ESCs) can form neuroectoderm (NE), providing a platform for in vitro dissection of NE formation. However, human ESCs can differentiate into all three germ layers. It thus is crucial to develop efficient methods for differentiation of human ESCs into NE cells. Both plating cell density and localized cell density (LCD) affect NE differentiation. Here, we developed a cell cluster-based NE differentiation method, in which both plating cell density and LCD are under control. Using our new method, high plating cell densities promote expression of PAX6, a NE marker protein. Two SMAD signaling blockers, SB431542 and NOGGIN, downregulate OCT4 and upregulate PAX6, while does not affect mRNA expression of GATA2 after 5 d of differentiation. Moreover, IB analysis showed a time-dependent upregulation of PAX6 and beta-III-tubulin together with a downregulation of OCT4 during the neural differentiation. Coexpression of both TH and beta-III-tubulin in the H9-derived cells was also detected, proving the NE cells have an ability to differentiate into one of the specific neurons. Together, we established a simple method for generating NE cells from H9 cells, which might contribute to develop high efficient method for neural differentiation.

OBJECTIVE

The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB)-derived progenitors, used in transplantation.

METHODS

We correlated gene expression patterns of highly enriched steady-state peripheral blood (PB)- and mPB-derived CD71+ cells by microarray and ingenuity pathway analyses, to identify the transcriptional program during in vitro erythroid differentiation.

RESULTS

The gene expression was more than doubled in mPB-derived (4180 genes) compared to PB-derived erythroid progenitors (1667 genes) while PB-and mPB-derived erythroid progenitors shared 1534 common genes. Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin, where we identified common 13 downregulated and 30 upregulated genes. The most significant genes in mPB-derived erythroid progenitors were P4HB, DDIA3, ARPC2 and ATP5G3. Regarding G-CSF stimulation the G-CSF receptor CSF2RB (1.1-fold) was linked via STAT3 to erythroid-specific ALAS2 (2.9-fold) and GATA2 (1.3-fold) factors, all upregulated in mPB-derived erythroid progenitors, coupled to common upregulated NUDC gene involved in the proliferation of erythroid cells.

CONCLUSIONS

This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources.

OBJECTIVE

To investigate the origin of hematopoietic progenitors contained in the stromal vascular fraction (SVF) of human adipose tissue.

METHODS

Tissue samples obtained from lipectomies were subjected to enzymatic digestion with collagenase to obtain a single-cell suspension. The centrifuged cell pellet, termed SVF, was separated immunomagnetically into CD45(+) and CD45(-) cells and cultured in serum-free medium containing hematopoietic cytokines. The freshly isolated and cultured cells were evaluated to determine their ability to form hematopoietic colony-forming units in clonogenic assays and for the expression of certain hematopoietic transcription factors by reverse transcription-polymerase chain reaction; the gene expression level was compared to that in CD34(+) hematopoietic progenitor cells from cord blood (CB) and adult peripheral blood (PB). To characterize erythroid progenitors, burst-forming units-erythroid (BFU-E) were developed in a semisolid medium under different culture conditions, and the hemoglobin composition and globin gene expression in the erythroid colonies were determined.

RESULTS

The transcription factors SCL/TAL1, RUNX1, RUNX2 and GATA2 were expressed in both the CD45(+) and CD45(-) SVF populations; however, in contrast to our observations in the CD34(+) cells from CB and adult PB, GATA1 was not detected. Nevertheless, GATA1 could be detected in the SVF cells after seven days in culture, whereas its expression was upregulated in the CB CD34(+) cells. The analysis of BFU-E-derived colonies revealed that virtually all erythroid cells produced by SVF cells expressed fetal hemoglobin, and the γ-globin mRNA levels ranged between those obtained in the adult- and neonatal-derived erythroid cells. Moreover, the SVF-derived erythroid cells synthesized similar levels of α- and β-globin mRNA, whereas the α-globin transcript levels were consistently higher those of β-globin in the cells derived from CB or PB CD34(+) cells. Furthermore, although the cellular distribution of hemoglobin in the erythroid cells derived from the CD34(+) cells obtained from hematopoietic tissues was dependent on the presence or absence of serum in the culture medium, this did not affect the SVF-derived erythroid cells.

CONCLUSIONS

Our results demonstrate that hematopoietic progenitors in SVF have molecular and functional features that differ from those exhibited by circulating progenitors, suggesting the possibility of a different origin.

Hemodynamic forces regulate many aspects of blood vessel disease and development, including susceptibility to atherosclerosis and remodeling of primary blood vessels into a mature vascular network. Vessels of the lymphatic circulatory system are also subjected to fluid flow-associated forces, but the molecular and cellular mechanisms by which these forces regulate the formation and maintenance of lymphatic vessels remain largely uncharacterized. This issue of the JCI includes two articles that begin to address how fluid flow influences lymphatic vessel development and function. Sweet et al. demonstrate that lymph flow is essential for the remodeling of primary lymphatic vessels, for ensuring the proper distribution of smooth muscle cells (SMCs), and for the development and maturation of lymphatic valves. Kazenwadel et al. show that flow-induced lymphatic valve development is initiated by the upregulation of GATA2, which has been linked to lymphedema in patients with Emberger syndrome. Together, these observations and future studies inspired by these results have potential to lead to the development of strategies for the treatment of lymphatic disorders.

Specific genetic deficiencies are a rare cause that should be included in the diagnostic algorithm of disseminated herpetic lesions. The aim of this article is to describe the ocular herpetic manifestations in a rare genetic disorder called GATA2 deficiency. We present the clinical case of a 26-year-old male with dendritic ulcers in his cornea, marrow aplasia and idiopathic chronic lymphedema. He was diagnosed with GATA2 deficiency. GATA2 gene is critical for the genesis and function of hematopoietic stem cells. Its deficiency can cause myelodysplastic syndromes, congenital lymphedema and severe viral infections. Our patient presented these three manifestations, added to a deletion in 20q12 that confirmed the suspicion of GATA2 mutation. A bone marrow transplant was suggested as definitive treatment. The corneal herpetic epithelial lesion was analogous to a regular hepetic keratitis with none stromal keratitis.

OBJECTIVE

The stem cell theory in the endometriosis provides an advanced avenue of targeting these cells as a novel therapy to eliminate endometriosis. In this regard, studies showed that lovastatin alters the cells from a stem-like state to more differentiated condition and reduces stemness. The aim of this study was to investigate whether lovastatin treatment could influence expression and methylation patterns of genes regulating differentiation of endometrial mesenchymal stem cells (eMSCs) such as BMP2, GATA2 and RUNX2 as well as eMSCs markers.

METHODS

In this experimental investigation, MSCs were isolated from endometrial and endometriotic tissues and treated with lovastatin and decitabin. To investigate the osteogenic and adipogenic differentiation of eMSCs treated with the different concentration of lovastatin and decitabin, BMP2, RUNX2 and GATA2 expressions were measured by real-time polymerase chain reaction (PCR). To determine involvement of DNA methylation in BMP2 and GATA2 gene regulations of eMSCs, we used quantitative Methylation Specific PCR (qMSP) for evaluation of the BMP2 promoter status and differentially methylated region of GATA2 exon 4.

RESULTS

In the present study, treatment with lovastatin increased expression of BMP2 and RUNX2 and induced BMP2 promoter demethylation. We also demonstrated that lovastatin treatment down-regulated GATA2 expression via inducing methylation. In addition, the results indicated that CD146 cell marker was decreased to 53% in response to lovastatin treatment compared to untreated group.

CONCLUSIONS

These findings indicated that lovastatin treatment could increase the differentiation of eMSCs toward osteogenic and adiogenic lineages, while it decreased expression of eMSCs markers and subsequently reduced the stemness.

Human pluripotent stem cells (hPSCs)/OP9 coculture system is a widely used hematopoietic differentiation approach. The limited understanding of this process leads to its low efficiency. Thus, we used single-cell qPCR to reveal the gene expression profiles of individual CD34+ cells from different stages of differentiation. According to the dynamic gene expression of hematopoietic transcription factors, we overexpressed specific hematopoietic transcription factors (Gata2, Lmo2, Etv2, ERG, and SCL) at an early stage of hematopoietic differentiation. After overexpression, we generated more CD34+ cells with normal expression level of CD43 and CD31, which are used to define various hematopoietic progenitors. Furthermore, these CD34+ cells possessed normal differentiation potency in colony-forming unit assays and normal gene expression profiles. In this study, we demonstrated that single-cell qPCR can provide guidance for optimization of hematopoietic differentiation and transient overexpression of selected hematopoietic transcription factors can enhance hematopoietic differentiation.