[Clinical Study of Ibrutinib Combined with Venetoclax Regimen in the Treatment of Relapsed/Refractory Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To investigate the clinical efficacy of ibrutinib combined with venetoclax in the treatment of relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), and to analyze the factors affecting efficacy and prognosis. METHODS: Clinical data of 62 R/R DLBCL patients admitted to our hospital from August 2017 to July 2022 were retrospectively analyzed. All patients were treated with ibrutinib combined with venetoclax. The clinical efficacy and drug safety were evaluated. The effects of clinical features on short-term efficacy and overall survival (OS) were analyzed. RESULTS: The objective response rate (ORR) of 62 patients was 48.39%. The extranodal lesions, intermediate-high/high risk of NCCN-IPI, intermediate-high/high risk of IPI, progression or recurrence time <12 months were the risk factors affecting the short-term efficacy of chemotherapy in R/R DLBCL patients (all P < 0.05). The most common adverse effect was neutropenia (75.19%), and the incidence of grade Ⅲ-Ⅳ neutropenia was 52.71%. The 1-year and 2-year OS rates of 62 patients were 48.51% and 31.56%, respectively, and the median OS time was 12 months. Multivariate analysis showed that objective remission after chemotherapy [HR =0.080 (95%CI: 0.028-0.235)] was a protective factor for OS in R/R DLBCL patients, and intermediate-high/high risk of NCCN-IPI [HR =4.828 (95%CI : 1.546-15.080)] was an independent risk factor affecting the prognosis of R/R DLBCL patients. CONCLUSION: Ibrutinib combined with venetoclax can be used as an effective treatment regimen for R/R DLBCL, and NCCN-IPI can be used as a prognostic indicator. Objective remission after chemotherapy is beneficial for R/R DLBCL patients to achieve better OS.

Downregulation of microRNA­25­3p inhibits the proliferation and promotes the apoptosis of multiple myeloma cells via targeting the PTEN/PI3K/AKT signaling pathway.

Numerous studies have confirmed that microRNAs (miRNAs or miRs) have important roles in cancer biogenesis and development including multiple myeloma (MM). MicroRNA­25­3p (miR­25­3p) has been proven to promote cancer progression, whereas its functions in MM has not yet been reported, at least to the best of our knowledge. Therefore, the present study aimed to investigate the function of miR­25­3p in MM and to identify the potential underlying mechanistic pathway. Herein, it was found that miR­25­3p expression was significantly increased in MM tissues and cell lines. The upregulation of miR­25­3p was closely associated with anemia, renal function impairment international staging system (ISS) staging and Durie­Salmon (D­S) staging. A high level of miR­25­3p was predictive of a poor prognosis of patients with MM. In vitro, the knockdown of miR­25­3p suppressed the proliferation and promoted the apoptosis of RPMI­8226 and U266 cells, while the overexpression of miR­25­3p exerted opposite effects. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a well­known tumor suppressor, was confirmed as a target of miR­25­3p in MM cells. Moreover, it was found that the PTEN expression levels were decreased, and inversely correlated with miR­25­3p expression levels in MM tissues. Further analyses revealed that the overexpression of PTEN exerted effects similar to those of miR­25­3p knockdown, whereas the knockdown of PTEN partially abolished the effects of miR­25­3p inhibitor on MM cells. Accompanied by PTEN induction, miR­25­3p promoted PI3K/AKT signaling pathway activation in MM cells. Collectively, these findings demonstrate critical roles for miR­25­3p in the pathogenesis of MM, and suggest that miR­25­3p may serve as a novel prognostic biomarker and therapeutic target of MM.

Oridonin effectively reverses the drug resistance of cisplatin involving induction of cell apoptosis and inhibition of MMP expression in human acute myeloid leukemia cells.

Cisplatin is the first generation platinum-based chemotherapy agent. However, the extensive application of cisplatin inevitably causes drug resistance, which is a major obstacle to cancer chemotherapy. Oridonin is a diterpenoid isolated from Rabdosia rubescens with potent anticancer activity. The aim of our study is to investigate the role of oridonin to reverse the cisplatin-resistance in human acute myeloid leukemia (AML) cells. The effect of oridonin on human AML cell proliferation was evaluated by MTT assay, cell migration and invasion were evaluated by transwell migration and invasion assays in cisplatin-resistant human AML cells. Furthermore, cell apoptosis was examined by flow cytometry. The inhibitive effect of oridonin in vivo was determined using xenografted nude mice. In addition, the expressions of MMP2 and MMP9 were detected by Western blot. There was a synergistic antitumor effect between cisplatin and oridonin on cisplatin-resistant human AML cells in vitro and in vivo. In addition, the combination of cisplatin and oridonin synergistically induced cell apoptosis. Furthermore, the combination treatment not only inhibited AML cell migration and invasion, but more significantly, decreased the expressions of MMP2 and MMP9 proteins. Our results suggest that the synergistic effect between both agents is likely to be driven by the inhibition of MMP expression and the resulting increased apoptosis.

[Ceramide participates in cell programmed death induced by Type II anti-CD20 mAb].

OBJECTIVE: To explore the exact mechanisms of programmed cell death (PCD) induced by Type II anti-CD20 mAb in CD20+ non-Hodgkin lymphoma (NHL) cells, and to provide theoretical basis for anti-tumor ability of new CD20 mAb.
 METHODS: After incubation with Rituximab (a Type I anti-CD20 mAb) and Tositumomab (a Type II anti-CD20 mAb), Raji cells were stained by annexin V & propidium iodide (PI). The ratio of programmed death cells were measured by two channel flow cytometry (FCM). Before the treatment of anti-CD20 mAbs, Raji cells was incubated with a caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK) and a dihydroceramide synthase inhibitor fumonisin B1 (FB1) for 30 minutes to assess their inhibitory effect on PCD. High performance liquid chromatography (HPLC) was utilized to compare the ratio of programmed death cells between the pretreatment group (treated by Rituximab and Tositumomab) and the non-pretreatment group. The anti-CD20 mAbs-treated Raji cells were collected, and the ceramide levels in the Raji cells in the different pretreatment groups were also examined by HPLC, and the inhibitory effect of FB1 on the changes of ceramide levels in the Raji cells was measured. The Raji cells were incubated with different concentration C2-ceramide, C2-Ceramide-induced PCD was also evaluated by annexin V & PI staining after 16 hours. 
 RESULTS: Tositumomab (10 µg/mL) but not Rituximab (10 µg/mL) can induce significant PCD (28.6±4.2)% in Raji cells, with significant difference (t=26.48, P<0.01), which cannot be blocked by Z-VAD-FMK with a concentration range from 10 to 30 µmol/L (F=3.01, P>0.05). The cellular ceramide levels in Raji cells were significantly elevated after the treatment of Tositumomab (t=28.48, P<0.01). C2-ceramide can significantly induce PCD in Raji cells in a dose-dependent manner with a concentration range from 5 to 40 µmol/L (F=2.71, P>0.05). The dihydroceramide synthase inhibitor FB1 can significantly inhibit the elevated cellular ceramide levels (F=20.18, P<0.01) and cell programmed death induced by Tositumomab (F=17.02, P<0.01).
 CONCLUSION: Type II but not Type I anti-CD20 mAbs can induce caspase independent PCD in CD20+ NHL cells through the elevation of cellular ceramide levels. The PCD is not associated with classic caspase pathway.

Association between tumor necrosis factor-α gene polymorphisms and diffuse large B-cell lymphoma in Chinese Han population: evidence from two center case-control study and a meta-analysis.

OBJECTIVES: The tumor necrosis factor-α (TNF-α) gene, which plays crucial roles in tumorigenesis, is reported to be an independent marker for cancer. This study aims to examine the association between the TNF-α G308A polymorphism and DLBCL risk based on the two center case-control studies and meta-analysis. METHODS: In the current study, we performed a two centers case-control study to investigate the effect of the TNF-α G308A polymorphism on DLBCL risk in Chinese Han population. A meta-analysis including 10 published datasets along with current dataset, including 111 comparisons containing 34,041 cases and 42,730 controls were enrolled, was next performed to further confirm the association after literature search was conducted and relevant studies were identified from PubMed, Embase, and Web of Science. RESULTS: The TNF-α -308A allele was associated with a significantly increased DLBCL risk in the two independent patient case-control studies and additionally for pooled analysis from the two sets (P<0.05 for both). The result of meta-analysis further demonstrated that the A allele of -308A was significantly correlated with DLBCL risk under the allelic model (OR=1.35, 95% CI=1.27-1.44) without heterogeneity by fixed-effects model analysis (Q=17.30, P=0.139). Moreover, sensitivity analysis supported the robustness of this meta-analysis. CONCLUSION: This study suggested that -308A polymorphism may be associated with the susceptibility of DLBCL in a Chinese population. The further meta-analysis provides additional evidence supporting the above result that the risk allele of the -308A polymorphism may increase DLBCL risk.

Sodium selenite inhibits leukemia HL-60 cell proliferation and induces cell apoptosis by enhancing the phosphorylation of JNK1 and increasing the expression of p21 and p27.

Selenium is an essential trace element and has shown chemopreventive or therapeutic activities on human solid cancers; however, whether it has anticancer effects on leukemia has not yet been elucidated. The present study was designed to determine the role of selenium on HL-60 human promyelocytic leukemia cells. We found that 100 nM of sodium selenite (Se) had no significant effects on cell proliferation, apoptosis and the cell cycle; however, a higher concentration of 250 nM of Se significantly inhibited cell proliferation, promoted apoptosis and induced cell cycle arrest at the S phase after 48 h of treatment (P<0.05), thus demonstrating the anticancer activities of selenium in leukemia. However, the decrease in c-Jun NH2-terminal kinase 1 (JNK1) expression by targeting JNK1 using small interfering RNA attenuated the inhibitory effects of Se on cell proliferation and the induction of apoptosis. Mechanistic studies showed that the anticancer activities of Se were associated with the enhanced phosphorylation of JNK1 and the increased expression of the cell cycle regulators, p21 and p27, as well as the downregulation of cyclin D1. Our data provide further evidence that the appropriate concentration of selenium has therapeutic potential in leukemia.

MicroRNA-23a expression in paraffin-embedded specimen correlates with overall survival of diffuse large B-cell lymphoma.

It has been demonstrated that aberrant expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. Recently, specific miRNAs may serve as potential biomarkers for the diagnosis and prognosis of various types of tumor. MiR-23a is known to play important role in the development of cancers and deregulated in various hematological malignancies. The aim of the present study is to explore miR-23a as potential diagnostic and/or prognostic marker of diffuse large B-cell lymphoma (DLBCL). We compared the expression level of miR-23a in DLBCL patients (n = 104) and reactive lymph nodes as controls (n = 28) from formalin-fixed, paraffin-embedded tissues using quantitative reverse transcription-polymerase chain reaction. The expression level of miR-23a was significantly higher in DLBCL patients than in controls (P = 0.001). No significant association was observed between the miR-23a expression level and clinical features such as age, gender, Ann Arbor stage, performance status, lactate dehydrogenase, extranodal sites and International Prognostic Index score (IPI). Kaplan-Meier analysis showed that higher expression level of miR-23a was significantly associated with a poor overall survival (OS) in DLBCL patients (log-rank test, P = 0.029), and multivariable Cox regression revealed the expression of miR-23a (adjusted P = 0.034) and IPI (adjusted P = 0.021) was independently associated with OS. To our knowledge, we provide here the first evidence that miR-23a may represent a diagnostic and prognostic marker for DLBCL. DLBCL patients with a high expression level of miR-23a had a shorter OS than patients with a lower expression level. Further investigation of the changes may be of prognostic significance in clinical practice.

Role of polymorphisms in BCL-2 and BAX genes in modulating the risk of developing non-Hodgkin lymphoma.

The aim of this study was to investigate whether polymorphisms of - 938C/A and Thr43Ala in the BCL-2 gene and G - 248A in the BAX gene are associated with the risk of developing non-Hodgkin lymphoma (NHL). We genotyped polymorphisms of - 938C/A and Thr43Ala in the BCL-2 gene and G-248A in the BAX gene among 424 patients with NHL and 446 controls. We found that the - 938AA genotype of the BCL2 gene was significantly associated with the risk of developing NHL (p < 0.001) and this genotype was associated with advanced stage (p = 0.01). Meanwhile, individuals having - 248AG + AA genotypes were significantly associated with an increased risk of NHL (p = 0.01), and these genotypes were associated with larger tumor size (p = 0.02). The present study demonstrated that the - 938AA genotype of the BCL-2 gene and - 248AG + AA genotype of the BAX gene may be susceptible genotypes for NHL. There appeared to be an impact of the BCL2 - 938AA genotype on advanced stage and - 248AG + AA genotypes on tumor size in NHL.

Protein expression and subcellular localization of familial acute myelogenous leukemia-related factor.

The present study was designed to evaluate the expression and subcellular distribution of the familial acute myelogenous leukemia-related factor (FAMLF). A 14-amino acid epitope of the predicted open reading frame of the FAMLF gene was identified using bioinformatics. This polypeptide was synthesized, conjugated to keyhole limpet hemocyanin and was subsequently used to produce antibodies. The antibody titer and specificity were characterized using ELISA and western blot assays, respectively. The antibody detected FAMLF protein expression in several human leukemia cell lines, bone marrow cells derived from one acute myeloid leukemia patient and one chronic myeloid leukemia patient, but not in bone marrow cells of healthy subjects. The FAMLF/GFP fusion protein was expressed in both the nucleus and the cytoplasm of transfected NIH3T3 cells. Our results demonstrate that the FAMLF gene is expressed in an AML patient but not in healthy controls, suggesting its association with AML.

Analysis of the expression levels of survivin and VEGF in patients with acute lymphoblastic leukemia.

The purpose of this study was to investigate the association between an inhibitor of apoptosis (survivin) and vascular endothelial growth factor (VEGF) expression in patients with acute lymphoblastic leukemia (ALL) prior to and following chemotherapy. Reverse transcription (RT)-PCR and western blotting were employed to analyze survivin and VEGF mRNA and protein expression. Moreover, the concentrations of survivin and VEGF were determined by enzyme-linked immunosorbent assay (ELISA). Our data revealed that survivin and VEGF were overexpressed in patients with ALL prior to treatment, while survivin levels were significantly decreased following treatment. In addition, there was a positive correlation between survivin and VEGF concentrations in plasma. In conclusion, analysis of survivin and VEGF expression levels may improve the clinical diagnosis and treatment of ALL.

[Packaging of lentivirus carrying gene hßc and overexpression of gene hßc in NB4 cells].

This study was aimed to overexpress gene hßc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hßc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hßc gene and the differentiation behaviour of NB4 cells. The targeted hßc gene was amplified by PCR from the cloned vector carrying ORF of hßc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hßc, named pLV-hßc. And the pLV-hßc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hßc gene was detected by Western blot. Then, the NB4 cells over-expressing hßc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hßc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hßc gene. The expression of CD11b was up-regulated in NB4-hßc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hßc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hßc to differentiate to neutrophils, but can not make them fully matured.