Minhang Zhou

Epigenetic silencing of microRNA-193a contributes to leukemogenesis in t(8;21) acute myeloid leukemia by activating the PTEN/PI3K signal pathway

t(8;21) is one of the most frequent chromosomal translocations occurring in acute myeloid leukemia (AML) and is considered the leukemia-initiating event. The biologic and clinical significance of microRNA dysregulation associated with AML1/ETO expressed in t(8;21) AML is unknown. Here, we show that AML1/ETO triggers the heterochromatic silencing of microRNA-193a (miR-193a) by binding at AML1-binding sites and recruiting chromatin-remodeling enzymes. Suppression of miR-193a expands the oncogenic activity of the fusion protein AML-ETO, because miR-193a represses the expression of multiple target genes, such as AML1/ETO, DNMT3a, HDAC3, KIT, CCND1, and MDM2 directly, and increases PTEN indirectly. Enhanced miR-193a levels induce G(1) arrest, apoptosis, and restore leukemic cell differentiation. Our study identifies miR-193a and PTEN as targets for AML1/ETO and provides evidence that links the epigenetic silencing of tumor suppressor genes miR-193a and PTEN to differentiation block of myeloid precursors. Our results indicated a feedback circuitry involving miR-193a and AML1/ETO/DNMTs/HDACs, cooperating with the PTEN/PI3K signaling pathway and contributing to leukemogenesis in vitro and in vivo, which can be successfully targeted by pharmacologic disruption of the AML1/ETO/DNMTs/HDACs complex or enhancement of miR-193a in t(8;21)-leukemias.

Low Dose Decitabine Treatment Induces CD80 Expression in Cancer Cells and Stimulates Tumor Specific Cytotoxic T Lymphocyte Responses

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8+, but not CD4+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

Establishment and application of real-time quantitative PCR for diagnosing invasive Aspergillosis via the blood in hematological patients: targeting a specific sequence of Aspergillus 28S-ITS2

BackgroundInvasive aspergillosis (IA) is an important cause of morbidity and mortality in immunocompromised individuals. This study was conducted to identify a desirable target DNA sequence for the diagnosis of aspergillosis using real-time quantitative polymerase chain reaction (qPCR).MethodsGenomic DNA was extracted from Aspergillus, Candida, and bacteria species, and qPCR was applied to validate a partial ribosomal DNA 28S-ITS2 sequence. Ethylenediaminetetraacetic acid-anticoagulated blood samples were collected from 72 febrile hematological patients, while total DNA was isolated from plasma and whole blood for the Aspergillus qPCR. The results were analyzed using a receiver operating characteristic curve. All cases were evaluated using the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) diagnostic criteria.ResultsUse of qPCR yielded positive results for 15 Aspergillus species but negative results for Candida species, bacterial strains, and human DNA. The limit of detection was one copy per microliter of DNA. Analytical sensitivity and specificity were six copies of DNA and 100%, respectively. The standard curve showed that qPCR was reliable for Aspergillus detection and that significantly more DNA copies were obtained from whole blood than from plasma (P < 0.001). At a cut-off value ≥ 25 copies/μL, the diagnostic sensitivity and specificity for IA using 28S-ITS2 qPCR were 90.9% and 73.4%, respectively.ConclusionsThe use of qPCR with whole blood to detect and verify the 28S-ITS2 sequence is a specific and useful way to diagnose IA.

Detection and clinical significance of gene rearrangements in Chinese patients with adult acute lymphoblastic leukemia

Abstract 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.

The cancer‑testis antigen NXF2 is activated by the hypomethylating agent decitabine in acute leukemia cells in vitro and in vivo

Cancer‑testis antigens (CTAs) are a group of tumor‑associated antigens restricted to male germ cells under normal physiological conditions. CTAs are expressed in certain types of tumors and thus are a novel target for immunotherapy. Nuclear RNA export factor 2 (NXF2) is a CTA of which the expression pattern, regulation and clinical significance are unclear. In the present study, following treatment with a demethylating agent, decitabine, NXF2 expression was detected in the majority of the NXF2‑negative acute leukemia cell lines, but not in healthy donor samples. This finding was confirmed by western blot analysis. Eight primary acute leukemia bone marrow samples were treated with decitabine in vitro, and results showed that NXF2 expression was significantly upregulated. In another nine acute myeloid leukemia or myelodysplastic syndrome patients, it was noted that the expression of NXF2 was upregulated in all patients following the first cycle of decitabine, which suggested that NXF2 was activated by decitabine treatment in vivo. Furthermore, NXF2 expression in acute leukemia cells was demonstrated to be regulated by CpG island hypermethylation. To the best of our knowledge, this is the first study to demonstrate that NXF2 is activated by demethylation in acute leukemia cells in vitro and in vivo. NXF2 may therefore serve as a novel target for immunotherapy against acute leukemia.

Clinical outcome of 30 patients with bone marrow metastases

Objective: Cancer patients with bone marrow metastases are rare and dismal. The study was to identify the clinical features and prognostic factors in cancer patients with bone marrow metastases. Patients and Methods: A total of 30 patients with bone marrow metastases were reviewed between September 2007 and September 2013. Bone marrow metastases were identified by bone marrow aspiration. Results: The median age was 56.5 years (range, 8–85 years). The two most common primary tumor sites were the stomach (7, 23.3%), breast (5, 16.7%). Bone metastases (27, 90.0%) were the most common concurrent metastases. The most common cause for bone marrow aspiration was anemia and thrombocytopenia (10, 33.3%). The median survival time was 3 months (range, 0.5–82 months). Patients with good performance status (n = 19) had a longer median survival time than patients with poor performance status (n = 11) (8 months vs. 1 months, P = 0.041). Patients with primary unknown origin (n = 5) had a significantly shorter overall survival time than patients with known origin (n = 25) (1 month vs. 6 months = 0.010). The median survival time was 9 months in the systemic therapy group (n = 21) and 1 month in the best supportive care group (n = 9) (P = 0.000). Conclusion: To make primary origin clear and start systemic antitumor therapy is beneficial for patients with bone marrow metastases.

The role of miR-92b in cholangiocarcinoma patients

Purpose: The role of microRNA (miRNA) in cholangiocarcinoma was not clear. The aim of this study was to find the potential diagnostic and prognostic miRNA in cholangiocarcinoma patients. Methods: The miRNA expression profiles in cholangiocarcinoma patients from The Cancer Genome Atlas and Gene Expression Omnibus (GSE53870) were analyzed. The comparison of overall survival was performed using the Kaplan–Meier method. The targeted genes of prognostic miRNA were identified in miRanda, PicTar, or TargetScan, and their cell signaling pathways were analyzed by the Database for Annotation, Visualization and Integrated Discovery. Results: In The Cancer Genome Atlas and the Gene Expression Omnibus miRNA dataset, miR-92b and miR-99a were found with concordant directionality, up-regulated and down-regulated, respectively. In The Cancer Genome Atlas survival data, patients with the high level of miR-99b had obviously shorter overall survival time (P=0.038). However, the level of miR-99a was not found to be significant. The 17 shared target genes of miR-92b were identified, such as DAB21IP, BCL21L11, SPHK2, PER2, and TSC1. The related pathways included positive regulation of transcription, positive regulation of cellular biosynthetic process, regulation of programmed cell death, etc. Conclusion: miR-92b was up-regulated in cholangiocarcinoma compared with normal controls. The high level of miR-92b was associated with adverse outcomes in cholangiocarcinoma patients, which might be partly explained by the targeted genes of miR-92b and their signaling pathways.

12- O -tetradecanoylphorbol-13-acetate (TPA) increases murine intestinal crypt stem cell survival following radiation injury

Radiation enteropathy is a common complication in cancer patients following radiation therapy. Thus, there is a need for agents that can protect the intestinal epithelium against radiation. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce differentiation and/or apoptosis in multiple cell lines and primary cells. In the current report, we studied the function of TPA in radiation induced enteropathy in cultured rat intestinal epithelial cell line IEC-6 after ionizing radiation (IR) and in mice after high dose total-body gamma-IR (TBI). In IEC-6 cells, there were reduced apoptosis and cell cycle arrest in TPA treated cells after IR. We detected a four-fold increase in crypt cell survival and a two-fold increase in animal survival post TBI in TPA treated mice. The beneficial effects of TPA were accompanied by upregulation of stem cells markers and higher level of proteins that are involved in PKC signaling pathway. In addition, TPA also decreased the TBI-augmented levels of the DNA damage indicators. The effects were only observed when TPA was given before irradiation. These results suggest that TPA has the ability to modulate intestinal crypt stem cells survival and this may represent a promising countermeasure against radiation induced enteropathy.

Susceptibility of Ph-positive all to TKI therapy associated with Bcr-Abl rearrangement patterns: a retrospective analysis.

Background Tyrosine kinase inhibitors (TKIs) have demonstrated success in the treatment of acute lymphoblastic leukemia (ALL) in patients that express BCR-ABL rearrangements (Philadelphia chromosome [Ph]). The current study aimed to assess the efficacy of TKIs and prognostic factors in the treatment of adults with Ph+-ALL. Methods In this multicenter retrospective study, the relationship between Ph+-ALL and treatment outcomes among Chinese patients receiving TKI-containing induction/consolidation chemotherapy was examined. A total of 86 Ph+-ALL patients were included and followed for 3.85 (0.43–9.30) years. Overall survival (OS) and event-free survival (EFS) were analyzed. Results A total of 86 Ph+-ALL patients (40 females and 46 males; median age: 34.0 years) were enrolled, including those with BCR/ABL transcripts 190 (n = 52), 210 (n = 25), and 230 (n = 2); BCR/ABL isoform determination was not available for 7 patients. Mortality was influenced by variable BCR/ABL transcripts and TKI administration, and BCR/ABL transcripts, hematopoietic stem cell transplantation (HSCT), and TKI administration were associated with the occurrence of events. The OS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKI administration (P = 0.008), the EFS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKIs (P = 0.012), and also higher than those with TKI salvage administration (P = 0.004). BCR/ABL transcripts 210 showed preferable OS and EFS compared with BCR/ABL transcripts 190 and 230 (P<0.05 for each). Conclusions The susceptibility of Ph+-ALL to TKI associated with the patterns of BCR-ABL rearrangement is demonstrated for the first time, thus adding another risk-stratifying molecular prognostic tool for the management of patients with Ph+-ALL.