[Using next generation sequencing technology to analyze gene mutations in patients with acute myeloid leukemia and the impact on prognosis].

Objective: To analyze the distribution of gene mutations in newly diagnosed acute myeloid leukemia (AML) patients, based on next generation sequencing technology (NGS) and to evaluate their value in AML risk stratification. Methods: The study analyzed 453 newly diagnosed AML(excluded acute promyelocytic leukemia, APL) patients from seven hospitals in Shanghai, from January 1st 2014 to December 31th 2017. RNA and DNA were extracted from pretreatment bone marrow mononuclear cells and targeted sequencing of AML genes were performed. The data of different groups was compared. Results: A total of 453 newly diagnosed AML patients were enrolled in the study, including 247 males and 206 females with a median age of 49.5 (range,11-85) years. A total of 540 mutations/fusion genes were detected in 289 patients, 29.1% (132/259) of whom with two or more mutations/fusion genes. In all patients, NPM1 was the most common mutation(12.8%), followed by ETO and TET2 mutation (11.92% and 11.04%, respectively) . And WT1 over-expression accounted for 10.6%. Patients over the age of 50 were with a higher frequency of mutations associated with epigenetic modification, 11.93% for ASXL1, 13.99% for DMNT3A, 6.58% for IDH1/IDH2, and 13.17% for TET2. The frequency of DMNT3A mutations was three times higher than that of patients under 50 years of age (P=0.017). In this study, a relatively low proportion of genetic mutations was observed in low-risk karyotype group. In the medium-risk karyotype group, the relatively high mutation frequencies were observed in NPM1, TET2, FLT3-ITD, DNMT3A, ASXL1, and CEBPA genes. In the poor-risk karyotype group, the mutation frequencies of ASXL1, TET2, DNMT3A and PHF6 genes were more than 10%, especially ASXL1 and PHF6 mutation frequencies were significantly higher than other molecular risk stratification groups (P<0.05). Of the 254 patients (56%) with normal karyotype AML (NK-AML), 56 patients were detected to have gene mutations about epigenetic modification. The median OS of this group was worse than that of patients without related mutations, while the median LFS had no significant difference. In patients with NK-AML older than 50 years, the OS and LFS of patients with epigenetic modification related gene mutations was 12 months and 10 months, versus 18 months and 12 months of patients without mutations. Conclusions: The gene mutations frequencies in AML patients with different age and molecular risk stratification groups are different. Epigenetics gene mutation frequencies, such as DNMT3A, ASXL1, IDH1/IDH2 and TET2,are higher in patients older than 50 years. A shorter OS can be observed in older patients(>50 years) with epigenetics gene mutation.

Paclitaxel inhibits the progression of cervical cancer by inhibiting autophagy via lncRNARP11-381N20.2.

OBJECTIVE: To observe the effect of paclitaxel on the autophagy of human cervical cancer cell lines by the expression regulation of lncRNARP11-381N20.2 as well as to explore the interaction and relationship between autophagy, proliferation, and apoptosis of cervical cancer cells. MATERIALS AND METHODS: Genome-wide expression profiles of tumors and their susceptibility to drugs were downloaded through TCGA database to find differentially expressed lncRNA RP11-381N20.2 in chemosensitive sensitive and insensitive groups. Expression of RP11-381N20.2 in 60 cervical cancer tissues and 30 normal tissues was detected by qRT-PCR. The relationship between the expression of RP11-381N20.2 and the clinicopathological parameters of lung cancer was statistically analyzed. The recombinant plasmid pcDNA-RP11-381N20.2 and pcDNA-NC of RP11-381N20.2 were transfected into SiHa cells by lipofectamine, respectively. The autophagy and phenotypic effects were observed. Cell proliferation was determined by colony formation assay. Apoptosis was detected by flow cytometry. Western blot was conducted to detect expressions of autophagy-related proteins. RESULTS: Genome-wide expression profiles of chemotherapy-sensitive and insensitive data in patients with cervical cancer in TCGA database were analyzed by edger package, results showed that the expression of lncRNA RP11-381N20.2 was significantly lower in the chemotherapy-insensitive group. qRT-PCR results showed that the expression of RP11-381N20.2 in cervical cancer was decreased, and the total survival time of patients was positively correlated with the expression of RP11-381N20.2. RP11-381N20.2 was associated with TNM (tumor node metastasis) staging and tumor size. Biological functions of SiHa cells showed that the expression of RP11-381N20.2 was negatively correlated with the treatment time and dose of paclitaxel. Colony formation assay showed that paclitaxel could inhibit the proliferation of cervical cancer cells in a dose-dependent manner. Flow cytometry showed that paclitaxel induced apoptosis of cervical cancer cells, which was more promoted after combination with RP11-381N20.2. Western blot results suggested that paclitaxel could induce autophagy in cervical cancer cells in a time- and dose-dependent manners. Paclitaxel combined with RP11-381N20.2 could significantly increase apoptosis of cervical cancer cells. CONCLUSIONS: During the killing process of paclitaxel on cervical cancer SiHa cells, cell autophagy would affect the efficacy, after overexpression of RP11-381N20.2 in SiHa cells, autophagy induced by paclitaxel was inhibited, thereby enhancing the killing effect of paclitaxel on tumor cells.

MicroRNA-574 enhances doxorubicin resistance through down-regulating SMAD4 in breast cancer cells.

OBJECTIVE: Drug resistance has become an important factor that threatens the survival and prognosis of patients with breast cancer, especially in patients with advanced breast cancer. Several microRNAs have been proved to participate in the resistant process; however, the role of miR-574 in doxorubicin (Dox) resistant breast cancer is still unclear. PATIENTS AND METHODS: Quantitative Real-time poly chain reaction (qRT-PCR) was employed to detect the expression level of miR-574 in breast cancer Dox-resistant MCF-7/Adr cell line and parental MCF-7 cell line. Using miR-574 mimics and inhibitors, miR-574 level was up- or down- regulated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was handled to detect the IC50, and flow cytometric analysis was employed to measure the apoptosis and cell circle. Dual-luciferase and Western-blot experiments were applied to verify the direct target gene of miR-574. RESULTS: miR-574 expression level was significantly higher in MCF-7/Adr cells compared to normal MCF-7 cells. Up-regulation of miR-574 level in MCF-7 cells promoted the cell growth and G0/G1-to-S phase transition but inhibited cell apoptosis. However, knockdown of miR-574 in MCF-7/Adr cells decreased the IC50 and cell growth. Using luciferase assay, SMAD4 was confirmed to be a potential target of miR-574, and the expression of SMAD4 protein was regulated by miR-574. In blood samples of patients, the miR-574 level before chemotherapy was higher than that after chemotherapy. CONCLUSIONS: We revealed miR-574 could promote doxorubicin resistance of breast cancer MCF-7 cells via down-regulating SMAD4, thus providing a novel target for advancing breast cancer chemotherapy.

Cisplatin suppresses tumor proliferation by inhibiting autophagy in ovarian cancer via long non-coding RNA RP11-135L22.1.

OBJECTIVE: To observe the effect of cisplatin-induced autophagy in human ovarian cancer cell lines and explore the correlation between RP11-135L22.1 with cisplatin-induced autophagy. MATERIALS AND METHODS: Genome-wide expression profile and chemotherapy sensitivity data of ovarian cancer were downloaded from TCGA database. It was found that the expression level of lncRNA RP11-135L22.1 differed between chemotherapy-sensitive group and insensitive group. Besides, RP11-135L22.1 expression levels were detected in 64 ovarian cancer tissues and 30 normal tissues by qRT-PCR. Relationship between RP11-135L22.1 expression levels in 64 ovarian cancer tissues and their clinicopathological characteristics were analyzed by x2-test. Cell viability was detected by CCK8 assay. Cell apoptosis and cell cycle were accessed by flow cytometry. HO8910 cells were selected for transfection of pcDNA-RP11-135L22.1, and qRT-PCR was used to evaluate RP11-135L22.1 expression in cisplatin-treated HO8910 cells. Western blot was performed to analyze the expression changes of autophagy-related proteins. RESULTS: Genome-wide expression profile of chemotherapy-sensitive and -insensitive patients with ovarian cancer from TCGA database was analyzed by edger package. It was found that RP11-135L22.1 level in chemotherapy-sensitive group was significantly lower than that of insensitive group. QRT-PCR results confirmed that RP11-135L22.1 was lowly expressed in ovarian cancer. The overall survival of patients was positively correlated with the expression of RP11-135L22.1. Furthermore, RP11-135L22.1 was associated with FIGO stage and tumor size. Flow cytometry showed that cisplatin could induce apoptosis and arrest cell cycle in ovarian cancer cells lines. CCK8 assay showed that cisplatin decreased viability of ovarian cancer cells. For in vitro study, HO8910 cells were cultured with medium containing different concentrations of cisplatin or treated with cisplatin for different times. The results revealed that RP11-135L22.1 expression was negatively correlated with the treating time and dose of cisplatin. Western blot showed that cisplatin induced autophagy in ovarian cancer cells in a time- and dose-dependent manner. Cisplatin combined with RP11-135L22.1 can reduce autophagy, increase the apoptosis and inhibit its activity of ovarian cancer cells to a certain extent. CONCLUSIONS: Cisplatin can induce autophagy in HO8910 ovarian cancer cells. After overexpression of RP11-135L22.1, it inhibited cisplatin-induced autophagy, thus enhancing the effect of cisplatin on ovarian cancer cells.

Ibrutinib presents antitumor activity in skin cancer and induces autophagy.

OBJECTIVE: Skin cancer is one of the most common malignancies in dermatology. Patient compliance and prognosis of skin cancer are poor. Ibrutinib, a Bruton's Tyrosine Kinase (BTK) inhibitor, is a new anticancer drug used to treat many cancers. Therefore, we aimed to explore the role of ibrutinib in the treatment of skin cancer. MATERIALS AND METHODS: Cell Counting Kit-8 (CCK8) and plate cloning assay were used to detect cell proliferation. Apoptosis was determined by flow cytometry. Western blotting analysis was used to analyze the expression of key proteins that regulated autophagy. Proliferation and apoptosis of skin cancer cells and induction of autophagy induced by ibrutinib were evaluated. RESULTS: CCK8 plate cloning assays showed that ibrutinib can gradually inhibit the skin cancer cell proliferation as the treatment time and dose increased. Results of flow cytometry showed that apoptosis in skin cancer cells were induced after ibrutinib treatment. Western blot showed that autophagy in skin cancer cells was found induced by ibrutinib and also related to the time and concentration of ibrutinib treatment. Combination treatment of ibrutinib and 3MA for skin cancer cells can significantly increase apoptosis. CONCLUSIONS: Ibrutinib has anti-tumor activity in skin cancer and can induce autophagy. Binding to autophagy inhibitors can promote ibrutinib's anti-skin cancer activity. Our experimental results provided new ideas for developing skin cancer drugs.

[Application of pegylated recombinant human granulocyte colony-stimulating factor to prevent chemotherapy-induced neutropenia in patients with lymphoma: a prospective, multicenter, open-label clinical trial].

Objective: To evaluate the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in prophylaxis neutropenia after chemotherapy in patients with lymphoma. Methods: This was a multicenter, single arm, open, phase Ⅳ clinical trial. Included 410 patients with lymphoma received multiple cycles of chemotherapy and PEG-rhG-CSF was administrated as prophylactic. The primary endpoint was the incidence of Ⅲ/Ⅳ grade neutropenia and febrile neutropenia (FN) after each chemotherapy cycle. Meanwhile the rate of antibiotics application during the whole period of chemotherapy was observed. Results: ①Among the 410 patients, 8 cases (1.95%) were contrary to the selected criteria, 35 cases (8.54%) lost, 19 cases (4.63%) experienced adverse events, 12 cases (2.93%) were eligible for the termination criteria, 15 cases (3.66%) develpoed disease progression or recurrence, thus the rest 321 cases (78.29%) were into the Per Protocol Set. ②During the first to fourth treatment cycles, the incidences of grade Ⅳ neutropenia after prophylactic use of PEG-rhG-CSF were 19.14% (49/256) , 12.5% (32/256) , 12.18% (24/197) , 13.61% (20/147) , respectively. The incidences of FN were 3.52% (9/256) , 0.39% (1/256) , 2.54% (5/197) , 2.04% (3/147) , respectively. After secondary prophylactic use of PEG-rhG-CSF, the incidences of Ⅳ grade neutropenia decreased from 61.54% (40/65) in the screening cycle to 16.92% (11/65) , 18.46% (12/65) and 20.75% (11/53) in 1-3 cycles, respectively. The incidences of FN decreased from 16.92% (11/65) in the screening cycle to 1.54% (1/65) , 4.62% (3/65) , 3.77% (2/53) in 1-3 cycles, respectively. ③The proportion of patients who received antibiotic therapy during the whole period of chemotherapy was 34.39% (141/410) . ④The incidence of adverse events associated with PEG-rhG-CSF was 4.63% (19/410) . The most common adverse events were bone pain[3.90% (16/410) ], fatigue (0.49%) and fever (0.24%) . Conclusion: During the chemotherapy in patients with lymphoma, the prophylactic use of PEG-rhG-CSF could effectively reduce the incidences of grade Ⅲ/Ⅳ neutropenia and FN, which ensures that patients with lymphoma receive standard-dose chemotherapy to improve its cure rate.

Placenta percreta with colon involvement in a twin pregnancy: case report and literature review.

A case of a woman with twin pregnancy having placenta percreta involving the colon, showed hematochezia symptoms, experienced bleeding which caused the patient's mortality. Placenta percreta with bowel involvement is a very serious complication of pregnancy. Symptoms are very atypical and it is very difficult to diagnose.

Ultracold fermi gases with resonant dipole-dipole interaction.

The superfluid phases in resonant dipolar Fermi gases are investigated by the standard mean-field theory. In contrast to the crossover from Bose-Einstein condensation (BEC) to Bardeen-Cooper-Schrieffer superfluid in Fermi gases with isotropic interactions, resonant dipolar interaction leads to two completely different BEC phases of the tight-binding Fermi molecules on both sides of the resonance, which are characterized by two order parameters with distinct internal symmetries. We point out that, near the resonances, the two competitive phases can coexist, and an emergent relative phase between the two order parameters spontaneously breaks time-reversal symmetry, which could be observed in momentum resolved rf spectroscopy.