地西他滨单药及联合半程和全程HA方案治疗骨髓增生异常综合征和急性髓系白血病的效果

目的:研究骨髓增生异常综合征和急性髓系白血病治疗中地西他滨单药及联合半程和全程HA方案的效果。方法:采用回顾性分析方法,选择2016年6月至2019年6月入院的80例骨髓增生异常综合征与急性髓系白血病患者,参考不同治疗方式分为研究组(40例)与对照组(40例),研究组选用地西他滨联合全程HA方案治疗,对照组选用地西他滨联合半程HA方案治疗;比较两组治疗后血小板、中性粒细胞减少持续时间、缓解率、总反应率及毒副反应发生率。结果:研究组治疗后血小板、中性粒细胞减少持续时间与缓解率、总反应率及毒副反应发生率依次为(10.18±0.98)d、(11.57±1.34)d、70.00%、82.50%、7.50%,对照组治疗后血小板、中性粒细胞减少持续时间与缓解率、总反应率及毒副反应发生率依次为(16.45±1.46)d、(18.03±1.92)d、30.00%、60.00%、25.00%。研究组较对照组治疗后血小板、中性粒细胞减少持续时间更短,缓解率及总反应率更高,毒副反应发生率更低(P<0.05)。结论:地西他滨联合全程HA方案治疗骨髓增生异常综合征和急性髓系白血病的效果优于地西他滨联合半程HA方案,且毒副反应少。     

Increased PRAME-Specific CTL Killing of Acute Myeloid Leukemia Cells by Either a Novel Histone Deacetylase Inhibitor Chidamide Alone or Combined Treatment with Decitabine

As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8⁺ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide in vitro. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201⁺ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME^100–108 or PRAME^300–309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.     

Differentially expressed microRNAs in the serum of cervical squamous cell carcinoma patients before and after surgery

Homoharringtonine (HHT), a plant alkaloid with antitumor properties originally identified nearly 40 years ago, has a unique mechanism of action by preventing the initial elongation step of protein synthesis. HHT has been used widely in China for the treatment of chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Omacetaxine, a semisynthetic form of HHT, with excellent bioavailability by the subcutaneous route, has recently been approved by FDA of the United States for the treatment of CML refractory to tyrosine kinase inhibitors. This review summarized preclinical and clinical development of HHT and omacetaxine for myeloid hematological malignancies.     

Advances in the treatment of acute myeloid leukemia: from chromosomal aberrations to biologically targeted therapy

We describe several recent advances in our understanding and treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) including the use of cytogenetics to classify these diseases and to identify therapies that are specific for the abnormalities. Cell lines have provided readily available and very relevant models to understand these diseases. The two clear successes include the use of retinoic acid for acute promyelocytic leukemia and tyrosine kinase inhibitors (e.g., imatinib) for chronic myelogenous leukemia. Very recent results suggest a particular activity of lenalidomide, an analogue of thalidomide, in MDS patients with deletions of the long arm of chromosome 5 (so-called 5q minus syndrome), and notable activity of azanucleoside DNA demethylating agents in MDS with loss of chromosome 7. However, for the vast majority of cytogenetic abnormalities found in AML/MDS, no specific therapies have been identified. The use of a variety of molecular biology techniques have identified a large number of genomic abnormalities; the challenge of the next several decades is to identify specific therapies for these molecular defects.     

Stem cell transplantation for leukemias following myelodysplastic syndromes or secondary to cytotoxic therapy

Two main forms of therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/AML) have been recognized. The most frequent type, occurring after treatment with alkylating agents, is characterized by abnormalities of chromosomes 5 and/or 7 and t-MDS/AML following treatment with topoisomerase II inhibitors and is associated with molecular aberrations of MLL (11q23) and AML-1 (21q22). Individuals with certain polymorphisms associated with impaired detoxification of cytotoxic agents have an increased risk of developing MDS or AML after treatment of unrelated cancers. Multidrug chemotherapy is less effective for patients with MDS, or AML following MDS, or t-MDS/AML when compared with primary AML, and results in lower complete remission (CR) rates and lower long-term survival. Patients with good risk cytogenetic features, such as t(15; 17), t(8; 21) and inversion 16 are an exception as their treatment outcome is comparable with primary AML patients. Patients who attain a polyclonal and/or a cytogenetic CR may be candidates for autologous stem cell transplantation. For the remaining patients, the only curative option is allogeneic stem cell transplantation with stem cells from a histocompatible sibling or an alternative donor. Reduced intensity conditioning regimens may be considered for patients older than 50 years or patients with comorbidities. The advice is to treat patients early after diagnosis and preferably before progression as these patients have the highest chance of a favorable outcome.     

Establishment and molecular characterization of decitabine‐resistant K562 cells

The clinical activity of decitabine (5‐aza‐2‐deoxycytidine, DAC), a hypomethylating agent, has been demonstrated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. However, secondary resistance to this agent often occurs during treatment and leads to treatment failure. It is important to clarify the mechanisms underlying the resistance for improving the efficacy. In this study, by gradually increasing concentration after a continuous induction of DAC, we established the DAC‐resistant K562 cell line (K562/DAC) from its parental cell line K562. The proliferation and survival rate of K562/DAC was significantly increased, whereas the apoptosis rate was remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA‐Seq. In addition, we also observed aberrant expression of DDX43/H19/miR‐186 axis (increased DDX43/H19 and decreased miR‐186) in K562/DAC cells. Ectopic expression of DDX43 in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC‐resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and DDX43/H19/miR‐186 may be involved in DAC resistance in K562.     

Identification of a common microdeletion cluster in 7q21.3 subband among patients with myeloid leukemia and myelodysplastic syndrome

Monosomy 7 and interstitial deletions in the long arm of chromosome 7 (−7/7q−) is a common nonrandom chromosomal abnormality found frequently in myeloid disorders including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML). Using a short probe-based microarray comparative genomic hybridization (mCGH) technology, we identified a common microdeletion cluster in 7q21.3 subband, which is adjacent to ‘hot deletion region’ thus far identified by conventional methods. This common microdeletion cluster contains three poorly characterized genes; Samd9, Samd9L, and a putative gene LOC253012, which we named Miki. Gene copy number assessment of three genes by real-time PCR revealed heterozygous deletion of these three genes in adult patients with AML and MDS at high frequency, in addition to JMML patients. Miki locates to mitotic spindles and centrosomes and downregulation of Miki by RNA interference induced abnormalities in mitosis and nuclear morphology, similar to myelodysplasia. In addition, a recent report indicated Samd9 as a tumor suppressor. These findings indicate the usefulness of the short probe-based CGH to detect microdeletions. The three genes located to 7q21.3 would be candidates for myeloid tumor-suppressor genes on 7q.     

Molecular genetics of chronic neutrophilic leukemia,chronic myelomonocytic leukemia and atypical chronic myeloid leukemia

According to the 2008 World Health Organization classification, chronic neutrophilic leukemia, chronic myelomonocytic leukemia and atypical chronic myeloid leukemia are rare diseases. The remarkable progress in our understanding of the molecular genetics of myeloproliferative neoplasms and myelodysplastic/myeloproliferative neoplasms has made it clear that there are some specific genetic abnormalities in these 3 rare diseases. At the same time, there is considerable overlap among these disorders at the molecular level. The various combinations of genetic abnormalities indicate a multi-step pathogenesis, which likely contributes to the marked clinical heterogeneity of these disorders. This review focuses on the current knowledge and challenges related to the molecular pathogenesis of chronic neutrophilic leukemia, chronic myelomonocytic leukemia and atypical chronic myeloid leukemia and relationships between molecular findings, clinical features and prognosis.     

Molecular mechanisms that produce secondary MDS/AML by RUNX1/AML1 point mutations

RUNX1/AML1 point mutations have been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. A heterozygous germline mutation of the RUNX1 gene causes a familial platelet disorder with a predisposition to AML. RUNX1 mutations have also been detected with high frequency in minimally differentiated AML M0 subtypes and myelodysplastic/myeloproliferative neoplasms. Here we propose a new disease category of myelodysplastic neoplasms (MDN) consisting of MDS refractory anemia with excess blasts and AML with myelodysplasia-related changes, including therapy-related cases. RUNX1 mutations have been detected in about 20% of patients with "MDN". Among the MDN cases, histories of radiation exposure, therapy-related myeloid neoplasms after successful treatment for acute promyelocytic leukemia, and leukemic transformation of myeloproliferative neoplasms have been reported to have a strong association with RUNX1 mutations. The mutations occur in a normal, a receptive, or a disease-committed hematopoietic stem cell. It is suspected that the "MDN" phenotypes are defined by the RUNX1 mutations in addition to some other abnormalities.     

Therapy-related myelodysplastic syndrome presenting as fulminant heart failure secondary to myeloid sarcoma

Rapidly progressive heart failure is commonly caused by an extensive myocardial infarction, a mechanical complication of infarction, myocarditis, or acute valvular insufficiency. We present an unusual case that was caused by a diffuse infiltration of the myocardium with leukemic cells (myeloid sarcoma). The patient presented with episodic shortness of breath, he was anemic and thrombocytopenic, and his bone marrow biopsy revealed myelodysplastic syndrome from treatment for oligodendroglioma. His clinical course was characterized by a chronic leak of cardiac enzymes, a new right bundle branch block, and a large pericardial effusion causing tamponade and death from fulminant heart failure and ventricular arrhythmias within 2 weeks. At autopsy, the heart was massively infiltrated with myeloblasts and other immature myeloid cells. There was no evidence of acute leukemia in the bone marrow or peripheral blood. Cardiac infiltration in a patient with myelodysplastic syndrome is extremely rare, especially in the absence of bone marrow involvement by blasts. The recognition of this entity is becoming increasingly important as the incidence of cardiac myeloid sarcoma may be on the rise as the number of patients receiving chemotherapy increases.     

The peculiarities of cytogenetic changes in different types of myelodysplastic syndrome

A cytogenetic study of bone marrow aspirate from 32 patients with different types of myelodysplastic syndrome (MDS) has been carried out. The patients were from eight regions of Ukraine. Chromosome deletions prevailed in the spectrum of karyotype changes. The largest number of chromosome abnormalities was revealed in patients with a refractory anemia with an excess of blasts (66.6% of cases). Chromosomal changes that involved three or more chromosomes occurred among 27% of all karyotype changes examined by us. Transformation of myelodysplastic syndrome to acute myeloid leukemia (AML) was found in 5 patients (45.4% of the cases) among 11 patients with abnormal karyotypes. We propose that cytogenetic confirmation of increased apoptosis in the bone marrow from the myelodysplastic syndrome patients is a phenomenon of chromosome fragmentation. The risk of transformation of myelodysplastic syndrome to acute myeloid leukemia was measured with the use of a new international score system, IPSS.     

The Bcl-2 inhibitor venetoclax inhibits Nrf2 antioxidant pathway activation induced by hypomethylating agents in AML

Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF-E2-related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT-199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine-increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase-1 and NADP-quinone oxidoreductase-1. Venetoclax treatment also leads to dissociation of B-cell lymphoma 2 from the Nrf2/Keap-1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.     

Characterization of in vitro and in vivo hypomethylating effects of decitabine in acute myeloid leukemia by a rapid, specific and sensitive LC-MS/MS method

DNA hypermethylation is a common finding in malignant cells and has been explored as a therapeutic target for hypomethylating agents (e.g., decitabine). Detection of changes in DNA methylation might serve as a pharmacodynamic endpoint to establish the biological activity of these agents and predict clinical response. We developed and validated a rapid, sensitive and specific LC-MS/MS method for determination of global DNA methylation (GDM) in vitro and in vivo. Ratios of 5-methyl-2′-deoxycytidine (5mdC) to the internal standard 2-deoxyguanosine (2dG) in mass signal were used to quantify GDM levels. The assay was validated in a linear range from 40 fmol to 200 pmol 5mdC. The intra-day precision values ranged from 2.8 to 9.9% and the inter-day values from 1.1 to 15.0%. The accuracy of the assay varied between 96.7 and 109.5%. This method was initially applied for characterization of decitabine-induced GDM changes in in-vitro-treated leukemia cells. Following exposure to 2.5μM decitabine, GDM decreased to ~50% of the baseline value. The clinical applicability of this method was then demonstrated in bone marrow samples from patients with acute myeloid leukemia treated with decitabine. Our data support the use of our LC-MS/MS method for clinical pharmacodynamic determination of changes in GDM in vivo.     

Correlations between cytogenetics and morphology in myelodysplastic syndromes

In order to detect possible relationships between cytogenetic abnormalities and morphologic features in myelodysplastic syndromes (MDS), 48 patients with MDS were investigated. Clonal cytogenetic abnormalities were present in bone marrow cells from 27 patients (56%). The most frequent single anomaly was del (5 q) (10 cases), followed by monosomy 7 (3 cases), trisomy 8 (3 cases) and del (20 q) (2 cases). Complex anomalies were present in 6 patients. Morphologically, according to the French-American-British (FAB) classification: 17 cases were considered as refractory anemia (RA), 17 as RA with excess of blasts (RAEB), 2 as RAEB in transformation, 2 as acquired idiopathic sideroblastic anemia and 10 as chronic myelomonocytic leukemia. With regard to the FAB classification, del (5 q) was often associated with RA and complex cytogenetic anomalies with RAEB. When myelodysplasia was studied in individual myeloid lineages, del (5 q) was associated with hypolobulated megakaryocytes, monosomy 7 with micromegakaryocytes and complex chromosomal anomalies with the association of two or more features of dysmegakaryocytopoiesis. Del (11 q) was associated with increased iron storage and del (20 q) with marked dyserythropoiesis. No correlation was observed between cytogenetic anomalies and features of dysgranulocytopoiesis.     

Acute promyelocytic leukemia with unusual karyotype

Acute myeloid leukemia (AML-M3) is associated with the translocation t(15;17)(q22;q12-21) which disrupts the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. We report a two-year-old patient with AML-M3 without the usual translocation t(15;17). Cytogenetic studies demonstrated normal appearance of chromosome 15 while the abnormal 17 homologue was apparently a derivative 17, der(17)(17qter-cen-q21:), the rearrangement distinctly shows deletion at 17q21 band and the morphology corresponding to an iso chromosome i(17q-). This case report is a rare cytogenetic presentation of acute promyelocytic leukemia (APML).     

A Comparison of Azacitidine and Decitabine Activities in Acute Myeloid Leukemia Cell Lines

Background

The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines.

Methodology/Principal Findings

Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 µM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping.

Conclusions/Significance

These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65∶35, RNA∶DNA.     

Recurrent DNMT3A R882 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome

Somatic mutations of DNMT3A gene have recently been reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We examined the entire coding sequences of DNMT3A gene by high-resolution melting analysis and sequencing in Chinese patients with myeloid malignancies. R882 mutations were found in 12/182 AML and in 4/51 MDS, but not in either 79 chronic myeloid leukemia (CML), or 57 myeloproliferative neoplasms (MPNs), or 4 chronic monomyelocytic leukemia. No other DNMT3A mutations were detected in all patients. R882 mutations were associated with old age and more frequently present in monoblastic leukemia (M4 and M5, 7/52) compared to other subtypes (5/130). Furthermore, 14/16 (86.6%) R882 mutations were observed in patients with normal karyotypes. The overall survival of mutated MDS patients was shorter than those without mutation (median 9 and 25 months, respectively). We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS, and may be an adverse prognostic event in MDS.     

Proliferating cell nuclear antigen (PCNA) and human malignant tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies

Lymphoma (Lymphocytic non-Hodgkin's malignant lymphoma) and leukemic (chronic lymphocytic, acute and chronic myeloid, myelomonocytic leukemia) cells were studied by indirect immunofluorescence to evaluate the presence of proliferating cell nuclear antigen (PCNA) and human malignant tumor nuclear antigen (HMTNA) in their nucleoli. Most cells in lymph node smears of lymphocytic non-Hodgkin's malignant lymphoma (NHML) developed a bright nucleolar fluorescence with HMTNA antibodies. PCNA was detected in nucleoli of a limited number of cells which apparently represent the proliferating cell population in these lymphomas. Similarly, in the bone marrow smears of patients with chronic lymphocytic leukemia most cells possessed a nucleolar fluorescence for HMTNA and PCNA was present in nucleoli of a limited number of cells. In the bone marrow smears of patients with myeloid or myelomonocytic leukemias most blastic or monocytoid cells also developed a bright nucleolar fluorescence with HMTNA antibodies and PCNA was present only in a small percentage of these cells. Leukemic cells with PCNA in their nucleoli like thekhuntigen might represent a proliferating cell population in late G1-early S phase.