[Inhibition of Autophagy Augments the Anticancer Activity of KPT-330 in Mantle Cell Lymphoma Cells].

OBJECTIVE: To investigate the influence of novel CRM1 inhibitor KPT-330 on the autophagy of mantle cell lymphoma (MCL) cells, and effect of KPT-330 on the prolifiration of MCL cells in the presence or absence of autophagy inhibitor. METHODS: CCK-8 assay was used to detect the effect of KPT-330 on MCL cell lines Z-138， Jeko-1， Granta-519， Rec-1. The effect of KPT-330 on autophagy features were determined by detecting acidic vesicular organelles (AVO) by MDC staining under fluorescence microscope and detecting protein expression of LC3B-II assessed by Western blot. Further combined application of lysosomal inhibitor Chloroquine (CQ) was used to observe its effect on the increase of LC3B-Ⅱ caused by KPT-330. CalcuSyn 2.0 software was used to detected the Combination index (CI) of KPT-330 combined with CQ. RESULTS: The proliferation of MCL cell lines （Z-138, Jeko-1, Grant-519, Rec-1） could be inhibited by KPT-330 in a dose-dependent manner (r =0.930, 0.946, 0.691, 0.968 respectively). The number of acidic vesicular organelles (AVO) and the expression of LC3B-II were increased in KPT-330 treated Jeko-1 and Granta-519 cells in a dose-dependent manner (r Jeko-1=0.993, r Granta-519=0.971). LC3B-II protein amounts still increased upon KPT-330 treatment with the existence of lysosomal inhibitor CQ in Jeko-1 and Granta-519 cells, which was higher than CQ or KPT-330 single drug group. The combination of KPT-330 and CQ produced the synergistic effects on cells proliferation inhibition with CalcuSyn 2.0 analysis. CONCLUSION: KPT-330 can inhibit MCL cell proliferation and induce autophagy. KPT-330 combined with autophagy inhibitor CQ could produce synergistic anti MCL effects, providing experimental basis for clinical combination therapy.

[Clinical Features and Outcomes of the Patients with B-Cell Chronic Lymphoproliferative Disease in the New Drug Era].

OBJECTIVE: To analyze the clinical characteristics of the patients with B-cell chronic lymphoproliferative disease（B-CLPD） in the new drug era and the effect of new drug treatment on efficacy and survival. METHODS: The clinical and laboratory data of 200 cases B-CLPD patients diagnosed between April 2015 and August 2021 were analyzed retrospectively. The clinical efficacy and survival of the patients under different treatments including Bruton tyrosine kinase（BTK） inhibitors， rituximab， and chemotherapy alone were analyzed. The prognostic factors affecting the survival of patients were analyzed by univarite analysis and multivariate analysis. RESULTS: There were 119 male（59.5％） and 81 female（40.5%） in 200 cases B-CLPD patients， the sex ratio(male/female) was 1.5∶1 with median age of 61（30- 91） years old. The distribution of subtypes were as fallows: 51 cases (25.5%) of chronic lymphocytic leukemia/small lymphocytic lymphoma（CLL/SLL）， 64（32.0%） cases of follicular lymphoma（FL）， 40（20.0%） cases mantle cell lymphoma（MCL）， 30（15.0%） cases of marginal zone lymphoma（MZL）， 10（5%） cases of lymphoplasmacytic lymphoma/waldenstrom macroglobulinemia（LPL/WM）， 5（2.5%） cases of B cell chronic lymphoproliferative disorders unclassified（B-CLPD-U） . The main clinical manifestation of 102 patients was lymph node enlargement, 32 cases were complicated with B symptoms. Among CLL/SLL patients， there were 12（23.5%） cases in Binet A and 39（76.5%） cases in Binet B/C. There were 29 patients（20.9%） in Ann Arbor or Lugano stage I-II and 110 cases（79.1%） in stage III-IV of other subtypes. The complete remission（CR） rate was 43.1%（25/58）， 40.2%（39/97）， 7.1%（1/14）， and overaIl response rate（ORR） was 87.9%（51/58）， 62.9%（61/97）， 28.6%（4/14） in the groups of BTK inhibitors， rituximab-based therapy， and chemotherapy alone. The 3-year OS rate and PFS rate in all patients was 79.2% and 72.4% respectively. The 3-year OS rate of patient with MZL, CLL/SLL, FL,WM was 94.7%, 87.7%, 86.8% and 83.3% respectively, while the 3-year OS rate of MCL was only 40.6%, which was significantly lower than other subtypes. The median OS of patients treated with BTK inhibitors and rituximab-based therapy was 20.5 and 18.5 months respectively， and the 3-year OS rate was 97.4% and 90.7%. However， the median PFS of patients receiving chemotherapy alone was 4 months， and the 1-year OS rate was 52.7%， which was statistically significant compared with the other two groups（P<0.05）. Univarite analysis showed that anemia， elevated lactate dehydrogenase， elevated ß2-microglobulin， and splenomegaly were the poor prognostic factors for OS（P<0.05）， elevated lactate dehydrogenase was also poor prognostic factors for PFS（P<0.05）. Multifactor analysis showed that anemia and elevated lactate dehydrogenase were the independent poor prognostic factors for survival（P<0.05）. CONCLUSION: The clinical features of B-CLPD was various， anemia and elevated lactate dehydrogenase are the prognostic factors for poor survival. BTK inhibitors and new immunotherapy can improve the survival and prognosis of patients in the new drug era.

[Expression and Clinical Significance of MiR-215 and KDM1B in Patients with Diffuse Large B Cell Lymphoma].

AbstractObjective:To investigate the expression of miR-215 and KDM1B in DLBCL patients, and to analysis its clinical significance. METHODS: Fifty patients with DLBCL treated in our hospital were selected as DLBCL group, and 30 cases of reactive proliferative lymphadenitis（RPL） were selected as controls. RQ-PCR was used to detect the expression level of miR-215, and immunohistochemistry was used to detect the expression of KDM1B protein. The expression of miR-215 and KDM1B in patients with different clinical characteristics and the survival rate of patients with different expression of miR-215 and KDM1B was compared. miR-215 mimics was transfected into SU-DHL-4 cells. Cell proliferation was detected by CCK-8. Cell apoptosis was measured by flow cytometry. The expression of KDM1B protein was detected by Western blot. RESULTS: The expression of miR-215 in DLBCL patients was significantly lower than that in control group, and the positive expression of KDM1B protein was higher, the difference was statistically significant(P<0.01). Spearman rank correlation analysis showed that the expression of miR-215 negatively correlated with KDM1B (r=-0.751，P<0.05). There was significant correlation of miR-215, KDM1B expression with symptoms of B，Serum level of LDH, International Prognostic Index(IPI), Ann Arbor stage,Tumor size, respectively in patients(P<0.05). Kaplan-Meier showed that 5-year overall survival rate of patients with high miR-215 expression was significantly longer than that with low miR-215 expression (P=0.013). The 5-year survival rate of the patients with high positive KDM1B expression was significantly lower than that with low positive expression(P=0.024). KDM1B protein was suppressed by the transfection of miR-215 mimics for 72 h, the cell proliferation rate in miR-215 mimics group was significantly lower than that in control group and NC mimics group (P<0.05), but cell apoptotic rate of miR-215 mimics were significantly higher(P<0.05). The expression of KDM1B protein was significantly lower than that in control and NC group(P<0.05). CONCLUSION: There are low expression of miR-215 and high expression of KDM1B protein in patients with DLBCL， suggesting that they may be the diagnostic and prognostic indicators of DLBCL. miR-215 can directly target KDM1B to inhibit cell growth and induce apoptosis.

[Rituximab plus Autologous Hemotopoietic Stem Cell Transplantation for The Treatment of CD5 Positive Diffuse Large B Cell Lymphoma with Autoimmune Hemolytic Anemia].

OBJECTIVE: To summarize the clinical features and therapy experience of a case of CD5 positive diffuse large B cell lymphoma (CD5+ DLBCL) with autoimmune hemolytic anemia (AIHA). METHODS: A 49-years old patient was investigated. The routine blood examination, bone marrow smear, Coombs test, serological test, chest CT, abdominal MR and immunochemistry etc were performed for this patient; and therapeutic effects of the chemotherapy regimen consisting of rituximab plus autologous hematopoietic stem cell transplantation (auto-HSCT) were observed. RESULTS: The cervical lymphnode biopsy confirmed CD5+ DLBCL; the severe anemia, reticulocyte increase, Coombs test positive, and erythroid hyperplasia in bone marrow all suggested the occurence of autoimmune hemolytic anemia (AIHA). After plasma exchange, immune suppression using methylprednisolone, blood transfusion, one course of chemotherapy with "R-CHOP-E", the symptoms of AIHA in patient disappeared. After a continuous treatment for 3 courses of "R-CHOP-E", the bone marrow infiltration appeared, which was assessed as "PD", then the treatment was changed to the "R-ESHAP" for 4 courses, the patient was reassessed as "CR". The patient subsequently underwent auto-HSCT, followed up for 6 months, patientis still "CR". CONCLUSION: The status of the CD5+ DLBCL patient with AIHA is severe, and the prognosis is poor. The curative effect of the chemotherapy regimen with rituximab plus auto-HSCT for this patien is well.

[Study on serum protein mass spectrometric characteristics of acute leukemia].

OBJECTIVE: To study the serum protein differential expression in acute leukemia patients and healthy control by differential protein mass spectrometry. METHODS: Serum proteins of 51 acute leukemia (AL) patients and 10 healthy donors were extracted from their peripheral blood. After removing high abundance protein, serum low abundance proteins were separated by two dimensional gel electrophoresis, the differences of serum proteins in AL patients and healthy human were identified. The protein spots with differential expression were cut out and then undergone bleaching, gel digestion and peptide extraction. The peptide mass fingerprint analysis was performed by using MALDI TOF/TOF MS. The protein database MSDB Masort retrieval program was used to evaluate the results. RESULTS: Using Student's t test,19 statistically significant abnormal expression proteins in the serum of AL patients were found compared with the healthy controls (P < 0.05). The expression of α1-trypsin inhibitor (P < 0.01), prealbumin (P < 0.01), trypsin inhibitor (P < 0.01), apolipoprotein E (P < 0.01) and apolipoprotein A-Ⅳ (P < 0.01) decreased, while retinol binding protein (P < 0.05), globin HP2 (P < 0.05), serum lectin (P < 0.05), H factor homologue protein (P < 0.05) and serum amyloid A1 (P < 0.01) increased. Further stratified analysis found that high serum lectin expression in AL patient resulted in poor outcomes. CONCLUSION: There are a variety of serum proteins with differential expression in peripheral blood of AL patients. The differential expression of serum lectin is related to the therapeutic effect. The differential expression of these proteins can be used as a new diagnosis marker or prognostic indicator for acute leukemia.

[Phenylhexyl isothiocyanate induces gene p15 re-expression by regulating histone methylation and DNA demethylation in Molt-4 cells].

This study was aimed to investigate the regulatory effect of phenylhexyl isothiocyanate (PHI) on methylation of histone H3K4, H3K9 and demethylation of p15 gene in acute leukemia cell line Molt-4, and to explore the possible mechanism inducing re-expression of silent gene. The methylation status of histone H3K4, H3K9 and the expression of P15 protein in the Molt-4 cells treated with PHI were detected by Western blot; the methylation status of p15 gene in the Molt-4 cells before and after treatment with PHI was determined by methylation specific polymerase chain reaction (MSP); the expression level of p15 gene mRNA in Molt-4 cells treated with PHI was assayed by semiquantitative reverse transcription-PCR. The results indicated that the PHI could increase methylation of histone H3K4 and decrease methylation of histone H3K9 in concentration-and time-dependent manners. After treatment of Molt-4 cells with PHI for 5 days, the methylation of p15 gene was reduced, the significant hypermethylation of p15 gene was reversed, the silenced p15 gene re-expressed; the expressions of p15 mRNA and P15 protein were enhanced in concentration-dependent manner. It is concluded that probably through specifically regulating the methylation level of histone H3K4 and H3K9, the PHI causes the changes of chromosome space structure and results in the demethylation of CPG island in p15 gene, thereby induces the re-expression of p15 gene which was silenced.

[Phenylhexyl isothiocyanate induces gene p15 demethylation by down-regulating DNA methyltransferases in Molt-4 cells].

This study is to investigate the effect of phenylhexyl isothiocyanate (PHI), which has been proved to be a novel histone deacetylase inhibitor (HDACi) recently, on gene p15 de novo expression in acute leukemia cell line Molt-4, and to further study its potential mechanism. Modified methylation specific PCR (MSP) was used to screen p15-M and p15-U mRNA. DNA methyltransferasel (DNMT1), 3A (DNMT3A), 3B (DNMT3B) and p15 mRNA were measured by RT-PCR. P15 protein was detected by Western blotting. Hypermethylation of gene p15 was reversed and activation transcription of gene p15 in Molt-4 was de novo after 5 days exposure to PHI in a concentration dependent manner. DNMT1 and DNMT3B were inhibited by exposure to PHI for 5 days (P < 0.05). Alteration of DNMT3A was not significant. It is showed that PHI could reverse hypermethylation of gene p15 and transcriptional activation of gene p15 is de novo by PHI. It may result from down-regulating DNA methyltransferases, DNMT1 and DNMT3B, or up-regulating the histone acetylation that allows chromatin unfolding and the accessibility of regulators for transcriptional activation in the p15 promoter.