[The study of gene mutations in unknown refractory viral infection and primary hemophagocytic lymphohistiocytosis].

OBJECTIVE: To study the type and corresponding clinical characteristics of primary hemophagocytic lymphohistiocytosis (HLH) associated immune gene mutations in the refractory virus infection or HLH of unknown causes. METHODS: From December 2009 to July 2010, the patients with refractory virus infection or HLH of unknown causes were screened for the primary HLH associated immune genes mutations by DNA sequence analysis, including PRF1, UNC13D, STX11, STXBP2, SH2D1A and XIAP. The clinical characteristics and outcomes were followed up. RESULTS: Totally 25 patients with refractory virus infection or HLH of unknown causes were investigated for the 6 genes and 13 cases were found carrying gene mutations, composing of 6 of PRF1 mutation, 3 of UNC13D, and each one of STX11, XIAP, SH2D1A and STXBP2, respectively. Among the 13 cases with gene mutations, 5 suffered from Epstein-Barr virus associated HLH (EBV-HLH), 1 human herpes virus 7 associated HLH (HHV7-HLH), 1 HLH without causes, 4 chronic activated EB virus infection (CAEBV) with 1 progressing to Hodgkin's lymphoma carrying abnormal chromosome of t(15;17) (q22;q25) and hyperdiploid, 2 EBV associated lymphoma. Among the other 12 patients without gene mutation, 4 suffered from EBV-HLH with 1 progressing to peripheral T lymphoma, 8 suffered from CAEBV. CONCLUSIONS: Primary HLH associated immune gene mutations are critical causes of refractory virus infection of unknown causes, most patients manifest as HLH, some cases appear in CAEBV and EBV associated lymphoma. DNA sequence analysis is helpful to early diagnosis and correct decision-making for treatment.

[Clinical Analysis of Treating EBV Infection Accompanied with GVHD after Allon-HSCT by EBV-Specific Cytotoxic T Lymphocytes].

OBJECTIVE: To investigate the efficiency and safety of treating Epstein-Barr virus (EBV) infection of acute graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) by EBV specific cytotoxic T lymphocytes (EBV-CTL). METHODS: The Clinical characteristics, therapeutic efficacy and safety of 12 patients with EBV infection treated by EBV-CTL infusion after allo-HSCT in Department of Hemahlogy of Aero Space Center Hospital between Jan 2015 and May 2017 were analyzed retrospectioely. RESULTS: Our of 12 cases received EBV-CTL infusion after transplantation, 9 did not received Rituximab therapy due to the active infection, 4 cases including 3 received Ritaximab progressed into posttransplantation lymphoroliferetive disease (PTLD). The median time of EBV infection was 47 (22-71) days, median time of antivirus therapy before tramplantation was 10 (8-33) days, median time of first CTL infusion was 59(34-86) days after transplatation. The 43 cases-time CTL infusion was performed smoothly, no related harmful evnts occoured, no progression of GVHD was observed. After the first course of infusion, complete remission (CR), Partial remssion (PR) and no remssion (NR) were obtained in 9, 1 and 2 patients respectively, the relapse was observed in 4 patients who then received the socond course of infusion and all reached CR, the patient in PR did not reathed CR finally and died of GVGD at 5 months after transpplantation . Only 1 out of 2 cases of NR obtained CR, another 1 still was in NR, and died of transplantation related infection at 5 months after transplantation. 4 cases of PTLD were all cared. CONCLUSION: Preliminary results of this study suggest that EBV-CTL infusion is safe for the EBV infection combined with acute GVHD after all-HSCT. However, a further larger scale clinical studies are needed to prove the efficiency.

STI571 combined with vincristine greatly suppressed the tumor formation of multidrug-resistant K562 cells in a human-nude mice xenograft model.

BACKGROUND: The development of the targeted signal transduction inhibitor STI571 has prompted us to treat chronic myeloid leukemia in different ways. Since STI571 may reverse multidrug-resistance of K562/MDR cells in vitro, we studied the effect of STI571 on multidrug-resistant K562 cells in vivo. METHODS: Multidrug-resistant human leukemia cell line K562-n/VCR expresses both bcr/abl fusion gene and multi-drug resistance (mdr1) gene. It is a vincristine resistant cell line subcloned from the vincristine (VCR) sensitive cell line K562-n induced by vincristine in vitro. K562-n and K562-n/VCR cells were inoculated subcutaneously into both sides of nude mice breast (5 x 106 cells/each) to establish a human leukemia xenograft model. The incidence and volume of tumor were observed. In the tumor-bearing nude mice, anti-tumor drugs vincristine, daunorubicin (DNR), STI571, and STI571 plus VCR for the treatment of mdr1 and bcr/abl double positive leukemia were studied respectively. RESULTS: The tumor incidence was 100% in the nude mice inoculated with either K562-n or K562-n/VCR. The transcription of the mdr1 gene and expression of P-gp were negative in K562-n cells but positive in K562-n/VCR cells. The intracellular accumulation of DNR in K562-n cells was higher than that in K562-n/VCR cells (P < 0.05). The tumor incidence of K562-n/VCR cells in nude mice was much higher than that of K562-n cells in chemotherapy groups, and the mean volume of the tumors was also larger (P < 0.05). STI571 combined with VCR significantly suppressed the proliferation of K562-n/VCR cells. CONCLUSIONS: The MDR characteristics of K562-n/VCR in vivo were the same as in vitro. STI571 had a significant tumor-suppressing effect on VCR-sensitive leukemia cells and a moderate effect on MDR leukemia cells. VCR combined with STI571 had an excellent tumor-suppressing effect on both K562-n/VCR and K562-n in the human-nude mice xenograft model.

[Spectrum of gene expression of a multi-drug resistant leukemia cell line with high tumorigenicity in nude mice].

OBJECTIVE: To investigate the mechanism of multi-drug resistance of K562-n/VCR cell line with both bcr-abl and mdr-1 expressions by clustering analysis of differential gene expression profiles. METHODS: By DNA microarray technique, genes differentially expressed by K562-n/VCR and K562-n cell lines were identified and analyzed. RESULTS: DNA microarray analysis of K562-n/VCR and K562-n cells was repeated three times and revealed 58 genes significantly differentially expressed among 12,800 genes arrayed. All but one was up-regulated in K562-n/VCR cells. The only gene down-regulated was a-myb. The up-regulated genes were MDR-associated genes, oncogenes, cytoskeleton, protein kinases and phosphatases, apoptotic and antiapoptotic factors, metabolism, transcriptional regulators associated with stress response, cell cycle checkpoint control, and genes for signal transduction proteins. CONCLUSION: These results indicate that, besides MDR-associated genes, other known and unknown genes may also be involved in the mechanism of multi-drug resistance.

[Synergistic inhibitory effect of STI571 in combination with arsenic trioxide on a multidrug-resistant leukemia cell line expressing bcr-abl].

OBJECTIVE: To study the synergistic effect of STI571, an inhibitor of tyrosine kinase in combination with arsenic trioxide (As(2)O(3)) on a multidrug-resistant leukemia cell line expressing bcr-abl. METHODS: The cytotoxic effect of STI571 alone or in combination with different concentrations of As(2)O(3) on both bcr-abl and mdr1 positive leukemia cell line K562-n/VCR was detected by MTT method. RESULTS: The cytotoxic effect of STI571 (1 micromol/L) combined with As(2)O(3) at concentrations 10(-5), 10(-6), 10(-7), 10(-8) mol/L (IC(50) 0.155 micromol/L) on K562-n/VCR cells was significantly higher than that of As(2)O(3) alone (IC(50) 1.879 micromol/L). The synergistic interaction on K562-n/VCR cells increased the cytotoxic effect by 12.1-fold. CONCLUSION: Combination of STI571 with As(2)O(3) has a synergistic inhibiting effect on leukemia cells expressing bcr-abl and mdr1.

[Clinical analysis of 12 cases of acute myeloid leukemia with Ph chromosome and BCR-ABL positive].

This study was purposed to analyze the characteristics of morphology, immunology, cytogenetic and molecular biology of leukemia cells in 12 AML patients with Ph(+) and their correlation with survival of patients. 12 patients with Ph(+) AML were diagnosed according to diagnostic criteria of WHO and existence of t(9;22) (q34;q11) or t(9;22) abnormality, meanwhile no evidence of CML chronic phase was observed. The results showed that 8 out of 12 cases were confirmedly diagnosed to be AML by morphologic and immunophenotypic examination, 4 cases were diagnosed as myeloid and B lymphocytic mixed acute leukemia. The Ph chromosome was detected in 10 cases by chromosome analysis at the first time of diagnosis, and some of the cases had coexistence of complex chromosome and/or normal karyotype. BCR-ABL transcript was detected in all 12 cases, including 7 cases with b3a2, 1 case with b2a2, 1 case with b2a2 variants, 2 cases with e1a2 and 1 case with e18a2. The 12 cases all got complete remission after chemotherapy and/or gleevec treatment, out of them 3 cases received chemotherapy and gleevec treatment, but 2 cases died; 9 cases received allogeneic hematopoietic stem-cell transplantation (allo-HSCT), 1 case died from relapse, among them 1 case died from transplant complications. The median survival was 24 (8 - 80) months, the overall survival of 3 years was (51.4 ± 17.7)%. It is concluded that the Ph(+) AML is a acute myelogenous leukemia with poor prognosis, but long-term survival may be achieved with HSCT as quick as after complete remission from gleevec and chemotherapy treatment. Meanwhile, the detection of BCR-ABL gene and it variants may be give more opportunity for diagnose and treatment, which can be used as routine screening for newly diagnosed leukemia.

[Predictable recurrence by regular monitoring minimal residual disease with flow cytometry in the patients with both AML and ALL: a single-center study of 163 cases].

OBJECTIVE: To study the predictable value of monitoring minimal residual disease (MRD) regularly by flow cytometry (FCM) in patients with acute leukemia (AL) in the first complete remission (CR(1)). METHODS: From April 2005 to July 2009, AL patients who had got CR(1) after chemotherapy were regularly monitored for MRD in bone marrow by FCM to relapse or to July 2010 in Beijing Daopei Hospital (not including those received stem cell transplantation). The special antibody combinations were employed for each patient according to aberrant expression of leukemia cells. MRD(+) was defined as the aberrant cells more than 0.01%. The probability of continuous CR (CCR) was calculated by Kaplan-Meier formula, and the statistical difference between two CCR probabilities was evaluated by log-rank test. RESULTS: A total of 163 AL patients in CR(1) were monitored to relapse or to July 2010. Among 89 AML patients referred to our hospital within 1 year after diagnosis, 30 cases were in MRD(+) and 59 cases MRD(-) till 12 months following chemotherapy, 3/30 patients in MRD(+) and 47/59 remained in CCR to July 2010. The probability of CCR at 24, 36 months was 13%, 13%in MRD(+) group, 94%, 78% in MRD(-) group respectively, the difference between them was statistically significant (P < 0.01). Among 35 ALL referred to our hospital within 5 months after diagnosis, 13 cases were MRD(+) and 22 cases MRD(-) till 5 months following chemotherapy, 0/13 patients in MRD(+) and 20/22 patients in MRD(-) remained in CCR to July 2010. The probability of CCR at 24, 36 months was 0% in MRD(+) group, 96%, 96% in MRD(-) group respectively, the difference between them was statistically significant (P < 0.01). Over the time point above, all patients with MRD(+) or their MRD from negative to positive relapsed finally, and most patients with MRD(-) remained CCR to July 2010. CONCLUSION: It had a clinical prognostic value to monitor MRD regularly by FCM in the patients with AL after CR(1).

[The clinical and laboratory features of 9 cases with gammadeltaT cell lymphoma or leukemia].

OBJECTIVE: To analyze the clinical and laboratory features of 9 cases of gammadeltaT cell lymphoma or leukemia. METHODS: From 2007 to 2011, 9 patients with gammadeltaT-cell lymphoma/leukemia were diagnosed in our hospital. The immunophenotype of the abnormal cells were detected by flow cytometry, clonal gene rearrangement of IgH, TCRgamma, TCRdelta by PCR, chromosome karyotype analysis by G banding, acute leukemia gene and the DNA of type 1 - 8 human herpes virus by multiple nested PCR, The gammadeltaT cells were determined by T cell with TCR gammadelta chain, the malignant gammadelta T cells by the abnormal expression of T cell antigens and the precursor malignant gammadelta T cells by the expression of CD34, TDT, CD99, CD1 a or acute leukemia genes. RESULTS: In the 9 patients with gammadeltaT cell lymphoma leukemia, significant malignant gammadeltaT cells infiltration of bone marrow were found in 8 with blast morphology. 5 were diagnosed as T-ALL/LBL (gammadeltaT type) and 4 HSgammadelta TCL. The clonal gene rearrangement of TCRgamma and/or TCRB were detected in 6/6 patients. Patients either did not achieve complete remission(CR) after induction therapy or relapsed quickly after CR. Only 4/5 patients remained continuous CR(CCR) at 2, 2, 3,12 months respectively, after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the fifth T-ALL (gammadeltaT) relapsed 1 month after allo-HSCT. CONCLUSIONS: The incidence of gammadelta T cell lymphoma or leukemia may be higher than reported, part of them were T-ALL/LBL with poor prognoses. FCM and clonal gene rearrangement of TCRgamma and/or TCRdelta are helpful to diagnosis. Allo-HSCT may be the only curative approach.

[Clinical and molecular biologic characteristics of 36 cases of leukemia with 11q23/mll].

This study was aimed to analyze the clinical and cytogenetic characteristics of acute leukemia with 11q23/mll rearrangement and explore the reasonable therapeutic principles. Characteristics in general situation, morphology, immunology, molecular biology, cytogenetics, treatment and overall survival of 36 cases of acute leukemias with mll gene rearrangement were studied and analyzed. The results showed that 36 cases with mll gene rearrangement were found positive (7.2%) in 494 patients with acute leukemia. Among the 36 cases of mll rearrangement positive, 32 cases were diagnosed as acute myeloid leukemia (AML) with myeloid antigen expression, of which 5 cases expressed lymphoblastic differentiation antigen; 4 cases were classified as B-lineage acute lymphoblastic leukemia (ALL), of which non-lineage myeloid expression pattern were found in 3 cases. In 29 out of 36 cases (80%) the clonal chromosomal aberration were detected, of which chromosome 11 aberration were observed in 22 cases. All patients received chemotherapy with a total response rate of 47.2%. Of the responded patients, 10 cases relapsed within 6 months, with a recurrence rate of 40%; 9 cases received hematopoietic stem cell transplantation (HSCT), 7 cases of which survived after transplantation. The median survival time of 36 cases was 16 months (range 2 - 46) and their 2-year overall survival rate was 41.4%. The 2-year overall survival rate of 9 patients who received HSCT was 87.5%. It is concluded that acute leukemia patients with mll gene rearrangement show poor response to chemotherapy, high recurrence rate and poor prognosis. Hematopoietic stem cell transplantation may be a reasonable treatment principle to improve these patients' survival situation.

[Establishment of a mouse model for detecting multi-drug resistant minimal residual leukemia cells expressing green fluorescent protein].

This study aimed to investigate the pathophysiology and therapy of multi-drug resistant model of minimal residual leukemia in mice. The multi-drug resistant model of minimal residual leukemia was established by using P388/VCR-G cell line expressing enhanced green fluorescent protein (EGFP) and DBA mice. The results showed that P388/VCR-G were inoculated in the abdominal cavities of DBA mice, the incidence of leukemia was 100%. Any of these mice with leukemia could not obtain remission spontaneously. The model of leukemia was sensitive to cyclophosphamide (Cy) and the time of survival was related to the dose of Cy received. The logarithm of cells inoculated in mice correlated regressionally with the dose of Cy. So this model was ideal for research on minimal residual leukemia. The distribution of residual leukemia cells in complete remission was not uniform in different organs including liver, spleen, thymus and bone marrow. Minimal residual leukemia cells could be found by fluorescent microscopy in freezing tissue slice. It is concluded that the multi-drug resistant model of minimal residual leukemia expressing EGFP can be established by using P388/VCR-G cell line and DBA mice. The minimal residual leukemia cells can be observed by fluorescence microscopy in complete remission stage.

[The reverse effect on drug-resistance against tyrosine kinase inhibitor STI571 in mdr1 and bcr-abl positive leukemic cells].

To explore the possibility of leukemia cell line of both bcr-abl and mdr-1 positive were cross-resistant to tyrosine kinase inhibitor STI571 and its reversal way, the inhibitory effect of STI571 on K562-n/VCR cells was detected with MTT method and reverse effects of CsA, TAM, IFN-alpha and CsA cominated with IFN-alpha were observed. The results showed that K562-n/VCR cell line expressing bcr-abl and mdr1 positive was resistant to STI571, and could be reversed by 5.18, 1.82 and 1.67-fold respectively, when treated with CsA, TAM, and IFN-alpha. It could be reversed by 34.87-fold with combination of half-dose CsA and IFN-alpha. In conclusion, amplification of mdr1 gene may contribute to drug-resistance of bcr-abl positive leukemic cells against STI571. The reversal agents, CsA, TAM and IFN-alpha show obviously reverse effects on drug-resistance. The combination of half-dose of both CsA and IFN-alpha display stronger effect than the full dose of either.