[Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice].

OBJECTIVE: The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. METHODS: Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. RESULTS: On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. CONCLUSION: Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

[Clinical Significance of Additional Chromosomal 8 Clonal Evolution in CML Progression].

OBJECTIVES: To explore the clinical significance of clonal evolution of additional chromosomal 8 in CML progression. METHODS: An unusual case with the clonal evolution from trisomy 8 to tetrasomy 8 accompanied by 2 time of CML blast crisis (BC) was reported. RESULTS: This patient suffered from 2 time of CML blast crisis and the additional chromosome 8 aberrations were accompanied. Trisomy 8 and tetrasomy 8 were detected at first CML blast crisis and second CML blast crisis, respectively. After tetrasomy 8 was developed, the c-Myc was over-expressed and the central nervous system leukemia happened in this case. Only high dose Ara-C and MTX regimen could induce remission for a short period. CONCLUSION: These findings suggested that additional chromosome 8 aberrations are important marker for poor prognosis of CML patients and contribute to a poor prognosis.

Relationship between Migration and HIV Risky Behavior: a Comparative Study of Returning Migrants and Non Migrants Based on Rural Out-of-school Youth in Jilin, China.

OBJECTIVE: To estimate the relationship between migration and HIV risky behavior when controlling for gender, age, and educational levels and to evaluate the gender differences in migration, HIV knowledge, and HIV risky behaviors among rural youth in China. METHODS: A cross-sectional, anonymous, investigative questionnaire for 1710 unmarried, out-of-school rural youth, aged between 15 and 24 years, was handed out in Gongzhuling county of Jilin province, China. RESULTS: 58.5% of participants had a history of migration, irrespective of gender. There were gender differences observed in other factors such as drug abuse (4.3% for males and 5.5% for females, P<0.01), multiple sexual partners (24.1% for males and 44.1% for females, P<0.01), and HIV knowledge rate (35.2% for males and 25.5% for females, P<0.001). While controlling for gender, age, and educational levels, the relationships between migration and drug abuse, selling sex, and non usage of condoms during last instance of sexual activity were found to be significant. The cases of premarital sex and multiple sexual partners were both not found to be related to migration. CONCLUSION: Among rural youth, the HIV risky behavior such as drug abuse, selling sex, and lack of condom use, is significantly related to migration, while premarital sex and multiple sexual partners seem unrelated to migration.

[Association between obesity and age at spermarche among Chinese Han boys aged 11-18 years].

OBJECTIVE: To analyze the association between obesity and age at spermarche among Chinese Han boys aged 11-18 years . METHODS: The height, weight and status of the spermarche of Chinese Han boys aged 11-18 years were selected from the data of 2010 National Physical Fitness and Health Surveillance.The body mass index (BMI), prevalence of spermarche in each age group and ages at spermarche by BMI groups were calculated. Chi square test was used to analyze the differences of prevalences of spermarche among the boys with different BMIs across ages. U-test was used to compare the differences of age at spermarche between the boys who were obese and not. RESULTS: In the boys aged 12 and 17 years in urban areas and boys aged 13 years in rural areas, the differences of prevalences of spermarche among the normal weight, overweight and obesity groups were significant (P<0.05). The age at spermarche in the obesity group (13.90 years) was 0.1 years earlier than that in the non-obesity group (14.00 years) (P<0.05). CONCLUSION: Obesity may make the age at spermarche ahead of time.

[Genetic characteristics and its prognostic significance for 217 adult patients with acute lymphoblastic leukemia].

OBJECTIVE: To evaluate cyto- and molecular genetic characteristics of adult patients with acute lymphoblastic leukemia (ALL) and its prognostic significance. METHODS: Two hundred and seventeen adult patients with ALL were analyzed for cyto- and molecular genetic characteristics with combined conventional cytogenetics, fluorescence in situ hybridization (FISH), real-time quantitative PCR (qPCR) and nested PCR. Significance of genetic findings for prognosis was evaluated. RESULTS: t(9;22)(q34;q11)/BCR-ABL has been the most frequent abnormality found in the cohort (56.3%). And 22.4% of cases with BCR-ABL detected by FISH was negative by cytogenetic analysis. Ratio of patients in high-risk group increased with age; Patients with B-ALL had a higher risk group than the average-risk group (98.40% vs. 65.70%, P=0.000). The overall survival (OS) rates at 3-month (67.30% vs. 85.10%, P=0.042), 6-month (55.1% vs. 80.4%, P=0.008), 12-month (34.0% vs. 59.1%, P=0.017) and 24-month (13.0% vs. 36.6%, P=0.010) were lower in high-risk group than in average-risk group, with medium OS time (11 months, 95% CI 8.0-13.9) being significantly shorter compared with the average-risk group (19 months, 95%CI 10.8-27.1). CONCLUSION: Adult patients with ALL have unique cyto- and molecular genetic characteristics, which has important value for prognosis and guiding treatment. Moreover, combined cytogenetic and molecular genetic techniques can precisely define sub-groups of ALL patients.

[Detection and clinical features of MLL gene rearrangement in adult patients with acute leukemia].

This study was purposed to investigate the incidence of mixed lineage leukemia (MLL) gene rearrangement and partner gene types as well as the clinical features and prognosis of acute leukemia (AL) with this rearrangement through detection in adult AL using combination of 3 techniques, and to evaluate the clinical value of this combination detection. The MLL gene rearrangement in 183 cases of adult AL was detected by combination of conventional cytogenetics, split signal FISH and multiplex nested PCR. The results showed that the incidence of MLL rearrangements in adult patients with AL was low (8.2%), and MLL-AF4 fusion gene was most common and predominant in acute lymphoblastic leukemia (ALL), while the MLL-AF6 and MLL-AF9 were most frequent in acute myeloid leukemia (AML). Extramedullary involvements were found in 40% of MLL-rearranged AL patients, and 33.3% of patients with MLL-rearranged AL reached to complete remission within 30 days during induction chemotherapy. In addition, in this cohort of MLL-rearranged adult AL patients, the 3-month relapse rate and 6-month overall survival rate were 50.0% and 50.0% respectively. It is concluded that the rate of missed diagnosis of CC technique for patients with MLL-rearranged AL reached to 60% in this study, while the combination of CC, FISH and multiplex nested PCR has been confirmed to have important significance for evaluating prognosis and conducting clinical therapy of patients with MLL-rearranged AL.

[Expression characteristics of isoforms of Ikaros and Helios in patients with leukemia and their mechanism].

This study was aimed to investigate the expression characteristics of two transcriptional factors in Ikaros family, Ikaros and Helios isoforms and their mechanism, as well as their correlation with clinical parameters, which play important roles in transcriptional regulation of hematopoiesis. Expression of Ikaros and Helios isoforms in a total of 163 patients with leukemia and correlations between Ikaros and Helios isoforms were analyzed by PCR. The results showed that different expression patters of Ikaros and Helios isoforms existed in leukemia patients, that is, Ikaros isoform (Ik-6) was predominantly expressed in acute lymphoblastic leukemia (ALL) with BCR/ABL fusion gene, while Helios isoform (He-i) was overexpressed in T-cell ALL patients. The results of cloning and sequencing demonstrated that the isoforms of Ikaros and Helios had different genetic alterations. The statistical correlation between these two isoforms not was found in this study, although interaction between Ikaros and Helios has been reported. It is concluded that although Ikaros and Helios belong to the same family with similar structure of zinc fingers, their isoforms have different expression profile, specific genetic alterations, and different clinical relevance in patients with leukemia. The connection and interaction between Ik-6 and He-i needs further research.

[Analysis of HIV/AIDS related risk behaviors and their impact factors among out-of-school adolescents in outflow areas].

OBJECTIVE: To analyze HIV/AIDS related risk behaviors among unmarried outside school adolescents and the impact factors in outflow areas, in order to provide basis for related health education. METHODS: Using cluster sampling method, we investigated a vocational training center for all 15- to 24-year-old unmarried outside school youths in one county. The structured questionnaire based on the Theory of Reasoned Action was anonymous, which filled envelopes on the spot. A total of 1 800 questionnaires were recovered, and 1 712 questionnaires were valid. Epidata 3.0 was used for establishing a database and SPSS 13.0 for statistical analysis. RESULTS: (1) The incidence of HIV/AIDS risk behaviors of the outside school adolescents was high: 18.0% of the respondents had sexual behavior, 27.3% had never used condom when sexed in the past three months, 31.0% had multiple sexual partners, and 9.7% had drug abuse experience; the rate of HIV/AIDS-related knowledge was only 25.1%; peer environment of respondents was poor. (2) The use of condoms was correlated with those who had higher score of AIDS knowledge, and who could talk about condoms in sexual intercourse; The multiple sexual partners' behavior was correlated with age, friends who were themselves multiple sexual partners, high score of the attitude, and the subjective norm; The commercial sex was correlated with the family address, high score of the HIV/AIDS-related knowledge, friends who had commercial sex, the subjective norm and the intention of behavior, The drug abuse behavior was correlated with age, high score of the HIV/AIDS-related knowledge, drug abuse among their friends, the subjective norm, and the intention of behavior. (3) Subjective norms and behavioral intentions could better predict the occurrence of HIV/AIDS risk behaviors. CONCLUSION: The outside school adolescents are at risk in lack of HIV/AIDS-related knowledge and coping skills of negative peer pressure, so providing the related health education before they go and work outside their home is the "critical period".

Combining nilotinib and imatinib improves the outcome of imatinib-resistant blast phase CML.

Imatinib resistance is an important hurdle in the treatment of chronic myeloid leukemia (CML), and CML patients with this drug resistance are often given a dismal prognosis. In this case report, an imatinib-refractory blast phase CML patient was treated with a combination of imatinib and nilotinib. A complete hematologic response was achieved within 3 months, the drug combination was well tolerated, and there was a relatively long bone-marrow complete remission. These results suggest that combining imatinib and nilotinib treatment may improve the outcome of imatinib-resistant CML patients in the blast phase. We hypothesize regarding the possible mechanism for the effectiveness of the drug combination by reviewing the recent literature.

A case report of a 33-year chronic phase survivor of chronic myeloid leukemia.

Here, we report a Philadelphia chromosome-positive chronic myeloid leukemia case with the longest chronic phase and overall survival to our knowledge ever reported in the medical literature. During the 33-year chronic phase, he was asymptomatic without any treatment and had normal blood cell values. BCR-ABL silencing might be referred to the uncommon long-term survivor.

[Clinical and biological features in refractory anemia with ringed sideroblasts with fluctuant platelet counts].

The objective of this study was to explore the differences between refractory anemia with ringed sideroblast (RARS) and RARS associated with marked thrombocytosis (RARS-T) in the clinical, biological features and prognosis. The morphological changes of cells were observed by bone marrow smear and biopsy. Immunologic phenotype was analyzed by flow cytometry, and chromosome was examined by conventional chromosomal analysis. JAK2 V617F and MPL W515L mutations were screened by allele-specific polymerase chain reaction (AS-PCR) and sequence analysis. The results showed that this case was clinically diagnosed as RARS with thrombophilia, the level of serum potassium was positively related with platelet counts. When platelets increased, the clusters of atypical giant platelets and megakaryocytes were observed in peripheral blood and bone marrow examined by bone marrow smear and bone marrow biopsy respectively, JAK2 V617F and MPL W515L mutations were negative. It is concluded that RARS may transform into RARS-T accompanied with megakaryocyte proliferation, large atypical platelets and negative JAK2 V617F. Preventing thrombophilia and monitoring relative gene mutations are necessary when atypical giant platelets and fluctuant platelet counts occurred in process of RARS with tendency to RARS-T.

[Apoptosis of NB4 cells induced by flavonoids of puerarin in vitro].

This study was aimed to investigate the effects of flavonoids of puerarin (PR) on apoptosis of acute promyelocytic leukemia (APL) cell line NB4 cells and its mechanism. The NB4 were treated with PR in vitro, the MTT assay was used to detect the inhibitory effect of PR on cell proliferation. The apoptosis of NB4 cells were detected by flow cytometry labelled with Annexin V/PI. The expressions of pml/rar alpha, bcl-2 and survivin were detected by real time reverse transcription-polymerase chain reaction (real time RT-PCR), the expressions of JNK, p38 MAPK, FasL, caspase 3, caspase 8 were detected by Western blot. The results showed that with the increasing of PR concentrations, the apoptosis rates of NB4 cells were gradually elevated. Simultaneously, the mRNA expression of pml/rar alpha, bcl-2 and survivin decreased, while the protein expression of JNK, FasL, caspase 3 and caspase 8 increased, which presented the positive correlation to PR concentrations. When PR combined with arsenic trioxide (ATO), the expression levels of above mentioned mRNA and protein decreased or increased more significantly. It is concluded that PR can effectively induce the apoptosis of NB4 cells. PR combined with ATO displays synergistic effect. It may be triggered by the activation of JNK signal pathway.

[Multiple myeloma cell line U266 apoptosis induced by velcade].

To investigate the effect of velcade on multiple myeloma cell line U266 apoptosis and its mechanism, cell viability was estimated by trypan blue dye exclusion. Annexin-V, mitochondrial transmembrane potential (delta psi m) and reactive oxygen species (ROS) labeled by DCFHDA were examined by flow cytometry, the expression of bcl-2 mRNA was detected by semi-quantitative RT-PCR. The results showed that the velcade inhibited the growth of U266 cells and reduced cell viability accompanied by appearance of morphologic characteristics of apoptosis. Velcade at 50 nmol/L increased Annexin V positivity and fluorescence intensity of DCF because of ROS generation while it decreased the delta psi m of U266 cells. Expression of anti-apoptotic gene bcl-2 mRNA also decreased. It is concluded that velcade inhibited the growth and reduce cell viability of U266 cells. Velcade can induce U266 cells apoptosis by intrinsic cell apoptotic pathway.

[ZAP-70 expression in 24 patients with B cell chronic lymphocytic leukemia].

To explore the ZAP-70 expression in chronic lymphocytic leukaemia (CLL) and its relationship with other prognostic factors, the expressions of ZAP-70 protein and CD38 in bone marrow or peripheral blood of 24 patients with B-CLL were determined by four-color flow cytometry. The results showed that ZAP-70 was positively expressed in 37.5% of all B-CLL patients, 20% (3/15) patients in Binet A stage, and 66.7% in Binet B + C. The expression of ZAP-70 had significant difference between Binet A and Binet B + C (P < 0.05). CD38 was expressed in 29.1% of B-CLL patients and 3 out of these cases were in stage A, 2 out of 3 cases in stage A co-expressed CD38 and ZAP-70. The expression of CD38 had no significant difference between Binet A and Binet B + C stage (P > 0.05). ZAP-70+ and CD38+ were both expressed in 83.3% of B-CLL patients (P < 0.05). It is concluded that ZAP-70 protein can be routinely measured by flow cytometry in the laboratory. High expression of ZAP-70 is correlated with other prognostic factors such as clinic stage, chromosome abnormalities and CD38 in B-CLL.

[Chronic myeloid leukemia onset with marked thrombocythemia].

This study was aimed to investigate the clinical, pathological and biological features of a special case of chronic myeloid leukemia (CML) with marked thrombocythemic onset. The morphological changes of cells were analyzed by using bone marrow smear and biopsy; Ph chromosome, a specific marker of CML, was assayed by conventional chromosomal analysis and fluorescence in situ hybridization, bcr/abl fusion gene was detected by reverse transcription-polymerase chain reaction. The results indicated that CML mimicked essential thrombocythemia (ET) at presentation was relatively rare and might be misdiagnosed as ET, bone marrow smear and biopsy revealed, marked thrombocytosis and moderate leukocytosis; RT-PCR, FISH and conventional chromosomal analysis demonstrated the existence of Ph chromosome and bcr/abl fusion gene. This special CML could progress into accelerated phase or blast crisis. The megakaryocytes in Ph+ ET were smaller than normal ones and had typically hypolobulated round nuclei. Patients diagnosed as Ph+ ET might progress into CML and showed a high tendency to myelofibrosis and blastic transformation. It is concluded that the value of routine cytogenetical and molecular biological analysis in diagnosis for potential CML cases, which mimicked ET as in this presentation, is very distinctive, and the importance is magnified by the recent availability of imatinib, a specific inhibitor of the bcr/abl tyrosine kinase produced by the Philadelphia chromosome. Every case of "ET" should be tested for the Philadelphia chromosome and bcr/abl transcript.

[JWA gene in regulating committed differentiation of HL-60 cells induced by ATRA, Ara-C and TPA].

The study was aimed to explore the role of gene JWA, a novel retinoic acid responsible and cytoskeleton associate gene, in regulating committed differentiation of HL-60 cell and the molecular mechanism in the course of differentiation and apoptosis of leukemic cells. By using FCM, the changes of CD13, CD14, CD15, CD11b and cell cycles were detected in HL-60 cells treated with ATRA (10(-6) mol/L), Ara-C (10 ng/ml) and TPA (10(-8) mol/L) respectively. The samples were determined by semi-quantitative reverse transcript-polymerase chain reaction (RT-PCR) and Western blot for the expression of JWA, Bcl-2, HSP27 and HSP70 at day 0, 2, 4, 6, 8. The results showed that HL-60 cells committedly differentiated into granulocyte-, monocyte-, macrophage-like cells. As a result, JWA was up-regulated in a time-dependent manner, while Bcl-2 was down- regulated at the same time. In ATRA and TPA group, the change of HSP70 had positive correlation with JWA, and negative correlation with Bcl-2. The expression of HSP27 was not detected. Contrast to the cells from APL patient, the expression of JWA need not be activated by ATRA in advance. In this study, we also exposed HL-60 cells in higher dose of Ara-C (20 ng/ml), and JWA expression underwent opposite trend comparing with in lower dose of Ara-C (10 ng/ml). It is concluded that JWA may play double important roles in regulating ATRA and TPA-induced differentiation and apoptosis in leukemic cells. The JWA expression had a negative correlation between induction and cytotoxic response. The difference of JWA expressions between HL-60 cell and ANLL patient cells would be involved in different leukemia pathogenesis.

[Influence of Qingdai compound on expression of bcr/abl and JWA in K562 cells].

To study the effects of Qingdai compound on proliferation and apoptosis of K562 cells, as well as the expression of bcr/abl and JWA mRNA, K562 cells were treated in culture with different concentrations of Qingdai compound (2.5, 5, 7.5, 10 and 20 mg/ml) and harvested at 24 hours. Then morphological changes were observed by light microscopy (LM); expressions of bcr/abl and JWA were detected with semi-quantitative RT-PCR. The results showed that morphological changes were observed as the increment of the Qingdai compound concentration. Inhibition effects on proliferation and apoptosis in K562 cells were seen. A concentration-dependent decreases were found in bcr-abl and JWA mRNA expression of K562 cells. Qingdai compound partially inhibited proliferation and induced apoptosis of K562 cells. Expressions of both bcr/abl and JWA, which took part in cell proliferation and apoptosis, were down-regulated in a dose dependent manner. In conclusion, Qingdai compound can partially inhibit the expressions of bcr/abl and JWA genes in K562 cells, and the clinical effect of Qingdai compound on CML may be associated with apoptosis of leukemic cells.

[Expression of P27(Kip1) and its clinical significance in acute and chronic leukemia].

To evaluate the expression of cyclin dependent kinase inhibitor P27(Kip1) in leukemia and to investigate its clinical significance, the P27(Kip1) protein in bone marrow or peripheral blood samples from 82 cases of leukemia was measured by Western blot and enhanced chemoluminescence (ECL). The results showed that the expression of P27(Kip1) protein in ALL was higher than that in ANLL (P = 0.033) and also that in CML (P = 0.008). P27(Kip1) expression in CLL was higher than that in CML too (P = 0.017). In acute leukemia, the effective rate (CR and PR) of initial chemical therapy in the group of P27(Kip1) > 0.655 was higher than that in the group of P27(Kip1) < or = 0.655, P = 0.041. For ANLL and ALL patients, the survival time in the group of P27(Kip1) > 0.655 was longer than that in the group of P27(Kip1) < or = 0.655, P = 0.0065. There were similar statistical significance for ANLL and ALL patients, P = 0.0271 and P = 0.0266 respectively. There was a negative correlation between chromosomal abnormalities and P27(Kip1) expression in ALL patients (r = -0.775, P = 0.04). The expression of P27(Kip1) protein appeared nothing to do with sex, age, white blood cell number, blast cell number in peripheral blood, serum LDH or uric acid. In conclusion, the expression level of P27(Kip1) protein is in relation to the effect of initial chemical therapy and survival time, so that the lower P27(Kip1) expression may associated with poor prognosis in acute leukemia.

[Human bone marrow mesenchymal stem cells express multiple hematopoietic growth factors].

To study the biological role of human cultured bone marrow mesenchymal stem cell (BM-MSC) in hematopoiesis by investigation of its expression of multiple hematopoietic growth factors, RT-PCR was used to analyze the expression of SCF, Flt3-ligand, TPO, LIF, G-CSF, GM-CSF, IL-3, IL-6 and IL-11 at mRNA level for human BM-MSC from healthy donors and patients with leukemia and lymphoma. BM-MSC were incubated with or without hydrocortison (HC). The results clearly showed that the cultured BM-MSC expressed mRNA of SCF, Flt3-ligand, TPO, LIF, IL-6 and IL-11 at passages 3 up to 15, but did not express G-CSF, GM-CSF and IL-3. The same expression pattern of above cytokines was seen also for the patient's BM-MSC. HC was able to induce BM-MSC to express G-CSF but not to express GM-CSF. BM-MSC seemed not to change morphologically after incubation with HC for up to 21 days. In conclusion, both normal and patient BM-MSC should be potential to promote hematopoiesis according to their expression of multiple hematopoietic cytokines, and HC is able to induce hematopoietic growth factor expression.