The value of routine bone marrow examination in patients with extranodal NK/T-cell lymphoma staged with PET/CT.

BACKGROUND: Limited evidence supports the omission of routine bone marrow (BM) examination (biopsy and aspiration) in patients with nasal-type extranodal NK/T-cell lymphoma (ENKTCL). This study was aimed at assessing whether BM examination provides valuable information for positron emission tomography/computed tomography (PET/CT)-based staging in this patient population. PATIENTS AND METHODS: Patients newly diagnosed with ENKTCL who underwent initial staging with both PET/CT and BM examination between 2013 and 2020 were retrospectively identified in two Chinese institutions. Overall, 742 patients were included; the BM examination was positive in 67 patients. RESULTS: Compared with BM biopsy alone, the combination of BM biopsy and aspiration assessment did not afford any additional diagnostic value. No patient with a positive BM biopsy was found to have early-stage disease by PET/CT. BM biopsy or PET/CT led to upstaging from stage III to IV as a result of BM involvement in 21 patients. In 135 patients with distant organ involvement, BM involvement was associated with worse overall survival (OS) and progression-free survival (PFS) compared with the corresponding durations in patients without BM involvement (2-year OS: 35.9% vs. 60.4%, p < .001; PFS: 26% vs. 40.7%, p = .003). No difference in survival was noted between groups judged positive based on PET/CT and BM biopsy. CONCLUSION: Compared with aspiration, BM biopsy led to the detection of more BM lesions. Baseline PET/CT can be safely used to exclude BM involvement in early-stage disease. Overall, routine BM examination affords diagnostic or prognostic value over PET/CT in patients with advanced-stage nasal-type ENKTCL.

Expression profiling of N6-methyladenosine modified circRNAs in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults, associated with poor prognosis and easy relapse of disease. Circular RNAs (circRNAs) were detected to be m6A modified and the role of m6A circRNAs has been reported in other diseases including cancers, however, their role has not been elucidated in AML yet. In the present study, we aimed to investigate the expression profiling of m6A circRNAs in AML. We performed m6A circRNAs microarray analysis to identify differentially expressed m6A circRNAs in bone marrow samples from AML patients and healthy individuals (control). Furthermore, bioinformatics analysis predicted the potential functions and relevant pathways that may be associated with the m6A circRNAs. The circRNA m6A methylation levels were found to be positively associated with the circRNAs expression, suggesting circRNA m6A modification could contribute to circRNA regulation in AML. Further analysis demonstrated that circRNA m6A modification might influence the circRNA-miRNA-mRNA co-expression network that may contribute to the circRNA regulatory network in AML. Our findings provide evidence of the differential expression profile of m6A circRNAs in AML, and circRNA m6A modification may contribute to circRNA regulatory function in AML.

Mutation profiling of circulating tumor DNA identifies distinct mutation patterns in non-Hodgkin lymphoma.

OBJECTIVE: Circulating tumor DNA (ctDNA) is emerging as a versatile biomarker for noninvasive genotyping and response monitoring in specific B-cell lymphomas; however, few studies have been conducted to explore ctDNA-based mutation profiling across non-Hodgkin lymphomas (NHLs) and genomic changes after initiation of chemotherapy. METHODS: A targeted sequencing of 362 genes was performed to detect the mutation profiles in paired blood and tissue samples from 42 NHL patients. Genomic alterations were explored in 11 diffuse large B-cell lymphoma (DLBCL) patients using paired blood samples collected pre- and post-R-CHOP chemotherapy. RESULTS: The frequencies of PIM1, MYD88, MYC, ZNF292, JAK, and MAF mutations were higher in aggressive than in indolent B-cell lymphoma and NK/T subtypes. Tumor mutation burden in blood samples was higher in aggressive than in indolent B-cell lymphomas and higher in patients who progressed than in those who responded to treatments. Our data also revealed significant enhance of concordance index through integrating mutated genes that were significantly associated with prognosis into International Prognostic Index-based prognostic model. Moreover, acquisition of mutations such as PCLO_p.L1220Tfs*3 was associated with resistance to R-CHOP in DLBCL patients. CONCLUSIONS: Our findings illustrated distinct mutation patterns across various NHL subtypes and suggested the association of genomic alterations in ctDNA with treatment outcomes.

Epigenetic silencing of E-cadherin gene induced by lncRNA MALAT-1 in acute myeloid leukaemia.

BACKGROUND: Epigenetic abnormalities in acute myeloid leukaemia provide us with a target for novel therapeutic strategies. The aim of the study was to verify the epigenetic regulatory mechanism of E-cadherin gene silencing induced by long non-coding RNA MALAT-1 in AML. METHODS: Expression of MALAT-1, E-cadherin, EZH2, SUZ12 and EED genes in AML patients was detected by RT-qPCR. After MALAT-1 silencing in AML cell lines, levels of the E-cadherin, EZH2, SUZ12, EED, DNMT1, DNMT3A and DNMT3B genes and encoded proteins were detected by RT-qPCR and Western blotting. The level of CpG island methylation and trimethylation modification of histone H3K27 in the promoter region of E-cadherin was detected by pyrosequencing and ChIP-qPCR. RIP-qPCR was used to detect the interaction between MALAT-1 and proteins. RESULTS: MALAT-1, EZH2 and EED gene expression was markedly increased in AML patients with E-cadherin down-regulation. A positive correlation between EZH2 or SUZ12 and MALAT-1 expression was observed. After MALAT-1 silencing, the expression of E-cadherin was up-regulated, whereas the expression of EZH2, SUZ12, DNMT1, DNMT3A and DNMT3B was down-regulated. Results of Western blotting were consistent with those of RT-qPCR. Methylation levels of E-cadherin in AML patients were higher than that in normal controls, which appeared to increase with age. Methylation of the CpG island and H3K27 trimethylation of E-cadherin were decreased after MALAT-1 silencing. RIP-qPCR suggested that MALAT-1 might be enriched by EZH2 and SUZ12. CONCLUSION: Our findings verified that MALAT-1 might lead to the transcriptional silencing of E-cadherin gene through the trimethylation of H3K27 mediated by recruiting EZH2 and SUZ12.

Identification of PLA2G7 as a novel biomarker of diffuse large B cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma is the most common form of non-Hodgkin lymphoma globally, and patients with relapsed or refractory DLBCL typically experience poor long-term outcomes. METHODS: Differentially expressed genes associated with DLBCL were identified using two GEO datasets in an effort to detect novel diagnostic or prognostic biomarkers of this cancer type, after which receiver operating characteristic curve analyses were conducted. Genes associated with DLBCL patient prognosis were additionally identified via WCGNA analyses of the TCGA database. The expression of PLA2G7 in DLBCL patient clinical samples was further assessed, and the functional role of this gene in DLBCL was assessed through in vitro and bioinformatics analyses. RESULTS: DLBCL-related DEGs were found to be most closely associated with immune responses, cell proliferation, and angiogenesis. WCGNA analyses revealed that PLA2G7 exhibited prognostic value in DLBCL patients, and the upregulation of this gene in DLBCL patient samples was subsequently validated. PLA2G7 was also found to be closely linked to tumor microenvironmental composition such that DLBCL patients expressing higher levels of this gene exhibited high local monocyte and gamma delta T cell levels. In vitro experiments also revealed that knocking down PLA2G7 expression was sufficient to impair the migration and proliferation of DLBCL cells while promoting their apoptotic death. Furthmore, the specific inhibitor of PLA2G7, darapladib, could noticeably restrained the DLBCL cell viability and induced apoptosis. CONCLUSIONS: PLA2G7 may represent an important diagnostic, prognostic, or therapeutic biomarker in patients with DLBCL.

Colorimetric immunosensor based on glassy carbon microspheres test strips for the detection of prostate-specific antigen.

Micro-sized glassy carbon microspheres (GCMs, typically 3 µm in diameter) instead of nano-sized gold nanoparticles (AuNPs, typically 20 nm in diameter) were for the first time used as signal markers for the quantitative detection of antigen such as prostate-specific antigen (PSA). After being treated with concentrated HNO3, GCMs bear carboxyl groups at their surfaces, which enables antibodies to be conjugated with GCMs to yield new type of micro-sized material-based colorimetric probes used for immunochromatographic test strips (ICTSs). The captured black GCMs (with strong and wide-band light absorption) on the T-line of ICTS were used both for qualitative and quantitative determination of PSA. In the case of quantitative determination, a lab-assembled optical strip reader system was used to measure the reflected LED light intensity at 550 nm. The sensing performances of the developed GCM-based ICTSs, such as sensitivity, selectivity, reproducibility, stability, and applicability, were investigated in detail. The developed GCM-based ICTSs can have much higher (3 times) detection sensitivity than AuNP-based ICTSs, showing promising applications in sensitive immunoassay.

[Oral Mucosal Diffuse Large B-cell Lymphoma Caused by Long-term Oral Methotrexate:Report of One Case].

A case of primary oral mucosal diffuse large B-cell lymphoma(DLBCL)due to long-term use of methotrexate(MTX)for the treatment of rheumatoid arthritis(RA)was admitted to the Department of Hematology,Fujian Medical University Union Hospital.We analyzed and discussed the clinical features,diagnosis and treatment,and prognosis of specific malignant lymphoma induced by MTX in this RA patient.Our purpose is to improve the awareness and knowledge of other iatrogenic immunodeficiency-associated lymphoproliferative disorders of clinicians and pathologists.This study provides a new reference for the clinical diagnosis and treatment of MTX-associated DLBCL.

Research progress in myosin light chain 9 in malignant tumors. / 肌球蛋白轻链9åœ¨æ¶æ€§è‚¿ç˜¤ä¸­çš„ç ”ç©¶è¿›å±•.

Myosin light chain 9 (MYL9) is a regulatory light chain of myosin, which plays an important role in various biological processes including cell contraction, proliferation and invasion. MYL9 expresses abnormally in several malignancies including lung cancer, breast cancer, prostate cancer, malignant melanoma and others, which is closely related to the poor prognosis, but the clinical significance for its expression varies with different types of cancer tissues. Further elucidating the molecular mechanism of MYL9 in various types of malignant tumor metastasis is of great significance for cancer prevention and treatment. At the same time, as a molecular marker and potential target, MYL9 may have great clinical value in the early diagnosis, prognosis prediction, and targeted treatment of malignant tumors.

The significance of chemokines in diffuse large B-cell lymphoma: a systematic review and future insights.

Despite the progress made in molecular and clinical research, patients with diffuse large B-cell lymphoma (DLBCL) still have a bad prognosis. Recently, chemokines/chemokine receptors have become the subject of interest in relation to DLBCL. Studies have demonstrated the important role of chemokines/chemokine receptors in the communication between DLBCL cells and tumor microenvironment. Studies have also reported the ability of chemokines/chemokine receptors in promoting the proliferation and invasion of DLBCL cells. Here, we summarize the data on mechanisms of DLBCL supporting the involvement of chemokine/chemokine receptor changes. We focus on the available evidence regarding chemokines/chemokine receptors as biomarkers and therapeutic targets for DLBCL.

[Clinical features and prognosis of children with mature B-cell non-Hodgkin's lymphoma: an analysis of 28 cases].

OBJECTIVE: To study the clinical features and treatment outcome of children with mature B-cell non-Hodgkin's lymphoma (B-NHL). METHODS: A total of 28 previously untreated children with mature B-NHL were enrolled and given the chemotherapy regimen of CCCG-B-NHL-2010. Among them, 20 were given rituximab in addition to chemotherapy. The children were followed up for 31 months (ranged 4-70 months). A retrospective analysis was performed for the clinical features of these children. The Kaplan-Meier method was used for survival analysis. A univariate analysis was performed to investigate the prognostic factors. RESULTS: Among the 28 children, 17 (61%) had Burkitt lymphoma, 8 (29%) had diffuse large B-cell lymphoma (DLBCL), and 3 (11%) had unclassifiable B-cell lymphoma. As for the initial symptom, 13 (46%) had cervical mass, 10 (36%) had maxillofacial mass, 9 (32%) had hepatosplenomegaly, 5 (18%) had abdominal mass, and 5 (18%) had exophthalmos. Of all children, 14 had a lactate dehydrogenase (LDH) level of <500 IU/L, 3 had a level of 500-1 000 IU/L, and 11 had a level of ≥ 1 000 IU/L. After two courses of chemotherapy, 21 children achieved complete remission and 7 achieved partial remission. At the end of follow-up, 24 achieved continuous complete remission and 4 experienced recurrence. The 2-year event-free survival rate was (85.7± 6.6)%. The children with bone marrow infiltration suggested by bone marrow biopsy, serum LDH ≥500 IU/L, and bone marrow tumor cells >25% had a low 2-year cumulative survival rate. CONCLUSIONS: The CCCG-B-NHL 2010 chemotherapy regimen combined with rituximab has a satisfactory effect in the treatment of children with B-NHL. Bone marrow infiltration on bone marrow biopsy is associated with poor prognosis.

Effects of Novel ncRNA Molecules, p15-piRNAs, on the Methylation of DNA and Histone H3 of the CDKN2B Promoter Region in U937 Cells.

Non-coding RNAs (ncRNAs) play key roles in epigenetic events. However, the exact mechanism of ncRNA guidance, particularly piwi-interacting RNAs (piRNAs), for the targeting of epigenetic regulatory factors to specific gene regions is unclear. Although piRNA function was first established in germ-line cells, piRNA may be crucial in cancer cells. This study investigated the potential roles of CDKN2B-related piRNA in leukemia cells to provide a potential tumorigenesis model of leukemia. CDKN2B-related piRNAs, hsa_piR_014637 and hsa_piR_011186 were transduced into the leukemia cell line U937 to study the effect of these two piRNAs on cell-cycle progression, apoptosis, heterochromatin formation, CDKN2B methylation and expression. Our results show that over-expressing hsa_piR_011186 promoted cell-cycle progression and decreased apoptosis. We also observed inhibition of CDKN2B gene expression. These effects were likely mediated by novel piRC (piRNA complex) of CDKN2B-related piRNA that associate with DNMT1, Suv39H1 and/or EZH2 proteins to modulate the methylation of DNA and histone H3 in the promoter region of the CDKN2B gene. The novel piRC complex facilitated epigenetic modifications on the promoter of cell-cycle regulating genes, providing an expanded view of the role of piRNA in the progression of leukemia cells.

Growth Inhibition Accompanied by MOB1 Upregulation in Human Acute Lymphoid Leukemia Cells by 3-Deazaneplanocin A.

Our purpose was to investigate the effect of 3-deazaneplanocin A (DZNep) on human T-cell acute lymphoid leukemia (T-ALL) cells, and to explore the underlying molecular mechanisms. The human T-ALL cell line Molt4 was treated with DZNep, and cell proliferation was examined. The expression of Mps one binder kinase activator 1 gene (MOB1) mRNA and protein was determined by RT-PCR and Western blotting, respectively. The histone modification effect of DZNep on the lysine 9 of histone 3 associated with MOB1 promoters was examined with chromatin immunoprecipitation and quantitative PCR, and CpG methylation in MOB1 promoters was detected by bisulfite sequencing PCR. DZNep treatment inhibited the growth of Molt4 cells. The expressions of MOB1 genes were upregulated by DZNep treatment, and histone methylations in their promoters were significantly reduced. The results indicate that DZNep is a promising therapeutic compound for the treatment of human T-ALL.

[The efficacy and safety of daptomycin for the treatment of Gram-positive bacterial infections in Chinese population].

OBJECTIVE: To investigate the efficacy and safety of daptomycin in Chinese patients with serious infections of Gram-positive coccus. METHODS: Clinical data were retrospectively collected from patients who were suspected with Gram-positive coccal infections and received daptomycin treatment between August 2010 and October 2012. RESULTS: A total of 203 Chinese patients from 26 centers were enrolled in our study, including 94 microbiologically diagnosed. Staphylococcus aureus was the most common pathogen (33%, 31/94) with 45.2% (14/31) methicillin-resistant Staphylococcus aureus (MRSA). According to the infection sites, primary bloodstream infection (45.8%, 93/203) was the most frequent, which was followed by skin and soft tissue infections (15.3%, 31/203). Seventy-seven cases (37.9%, 77/203) had bloodstream infections complicated with other infections (37.9%, 77/203), 13 of which were endocarditis. The clinical efficacy of intention-to-treatment (ITT) and modified ITT (MITT) analysis were 70.44% (143/203) and 78.72% (74/94), respectively. Seven patients (3.4%) represented drug-related adverse effect, but no serious adverse effect was reported. Moderate creatine phosphate kinase (CPK) elevation was observed in 4 patients (2%), which returned to normal range after drug withdrawl. CONCLUSION: Daptomycin is effective and safe for Chinese patients with serious infections of Gram-positive cocci. (registration number NCT10212601).

[Hypermethylation of CpG island of DLC-1 gene and arsenic trioxide-induced DLC-1 gene demethylation in multiple myeloma].

OBJECTIVE: To explore the role of hypemethylation of DLC-1 gene in the pathogenesis of multiple myeloma (MM) and examine the effects of arsenic trioxide (As(2)O(3))-induced demethylation of DLC-1 gene in U266 cell line. METHODS: The methylation status of DLC-1 gene was detected by methylation specific PCR (MSP) in MM patients from 2008 to 2012. And the expression of DLC-1 gene mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). Methylation statuses of DLC-1 gene exposed to As(2)O(3) were detected by bisulfite sequencing PCR (BSP). And the mRNA expressions of DLC-1 and DNA methyltransferase (DNMT1, T3a and 3b) were determined by real-time fluorescence quantitative PCR (RTFQ-PCR). RESULTS: Hypermethylation of CpG island of DLC-1 gene was observed in 37/52 (71.15%) MM patients. DLC-1 gene was not expressed after methylation. As(2)O(3) could induce DLC-1 gene demethylation. After 72-houe treatments of 0.5, 1.0 and 2.0 µmol/L As(2)O(3), the methylation rate of DLC-1 gene dropped from 95.38% to 63.07%, 30.00% and 7.69%. As compared with the untreated group, the expression of DLC-1 gene mRNA increased to (1.60 ± 0.09), (3.66 ± 0.17) and (5.29 ± 0.15) folds after exposures (all P < 0.05) . And As(2)O(3) could induce the expression of DNMT1, DNMT3a, DNMT3b gene mRNA (all P < 0.05). CONCLUSIONS: Methylation of DLC-1 gene is essential in the pathogenesis of MM and may provide a new diagnostic technique and drug target for the treatment of MM. And As(2)O(3) may activate the expression of DLC-1 gene through demethylation.

LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization.

OBJECTIVE: YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors; differentiating between these roles may depend on the YAP1 phosphorylation pattern. The specific function of YAP1 in B cell acute lymphoblastic leukemia (B-ALL), however, is currently unclear. Thus, in the present study, the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets. METHODS: The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting, quantitative real-time polymerase chain reaction, flow cytometry, immunostaining, and nude mouse subcutaneous tumorigenesis experiments. Gene expression levels of Hippo pathway-related molecules before and after verteporfin (VP) treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells. RESULTS: Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels. YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells; YAP1 was distributed in the nuclei in NALM6 cells. Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase. Before and after VP treatment, the expression of the upstream gene LATS1 was upregulated; its overexpression promoted YAP1-Ser127 phosphorylation. Further, YAP1 was distributed in the plasma. CONCLUSION: LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function; thus, VP, which targets this axis, may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

A lasso and random forest model using flow cytometry data identifies primary myelofibrosis.

Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34+ cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34+CD19+cells and CD34+CD38- cells on CD34+cells, CD13dim+CD11b- cells in granulocytes, CD38str+CD19+/-plasma, and CD123+HLA-DR-basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34+CD38- on CD34+ cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.

[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.

[Experimental Study on the Mechanism of Mangiferin Inhibiting Malignant Biological Characteristics of Multiple Myeloma and Exerting Anticancer Effect].

OBJECTIVE: To investigate the effect of pure Chinese herbal extract Mangiferin on the malignant biological behaviors of multiple myeloma (MM) cells, and to analyze the molecular mechanism of the anti-myeloma effect of Mangiferin, so as to provide experimental basis for MM replacement therapy. METHODS: U266 and RPMI8226 of human MM cell lines were intervened with different concentrations of Mangiferin. Cell proliferation was detected by CCK-8 method. Annexin V/PI double staining flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of apoptosis and related signaling pathway proteins, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of matrix metalloproteinase (MMP) and CXC chemokine receptor (CXCR) family. RESULTS: Mangiferin could inhibit the proliferation activity of U266 and RPMI8226 cells and induce cells apoptosis. After Mangiferin intervened in U266, RPMI8226 cells for 48 h, the expression of Bcl-2 family pro-apoptotic protein Bax was up-regulated, while the expression of survivin and Bcl-xL proteins was down-regulated and caspase-3 was hydrolyzed and activated to promote cell apoptosis, besides, the expression of Bcl-2 protein in U266 cells was also significantly down-regulated to induce apoptosis (P<0.05). After Mangiferin intervenes in MM cells, it can not only increase the expression level of tumor suppressor p53, but also induce programmed cell death of MM cells by inhibiting the expression of anti-apoptotic molecules and down-regulating the phosphorylation levels of AKT and NF-κB. In addition, after the intervention of Mangiferin, the expressions of CXCR4, MMP2 and MMP9 in U266 cells were down-regulated (P<0.05), while there is no effect on the expressions of CXCR2, CXCR7 and MMP13 (P>0.05). However, the expressions of CXCR4, MMP9, and MMP13 in RPMI8226 cells were down-regulated (P<0.01), the expression of MMP2 was weakly affected, and the expression of CXCR2 and CXCR7 was basically not affected (P>0.05). CONCLUSION: Mangiferin can inhibit the proliferation and induce apoptosis of MM cells, and its mechanism may be related to inhibiting the activation of NF-κB signaling pathway, affecting the expression of Bcl-2 family proteins, and inhibiting the expression of core members of MMP and CXCR family.

[The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia].

OBJECTIVE: To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML. METHODS: Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05). CONCLUSION: Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.

CD26lowPD-1+ CD8 T cells are terminally exhausted and associated with leukemia progression in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Novel effective treatment is an urgent unmet need. Immunotherapy targeting T cell exhaustion by blocking inhibitory pathways, such as PD-1, is promising in cancer treatment. However, results from clinical studies applying PD-1 blockade to AML patients are largely disappointing. AML is highly heterogeneous. Identification of additional immune regulatory pathways and defining predictive biomarkers for treatment response are crucial to optimize the strategy. CD26 is a marker of T cell activation and involved in multiple immune processes. Here, we performed comprehensive phenotypic and functional analyses on the blood samples collected from AML patients and discovered that CD26lowPD-1+ CD8 T cells were associated with AML progression. Specifically, the percentage of this cell fraction was significantly higher in patients with newly diagnosed AML compared to that in patients achieved completed remission or healthy controls. Our subsequent studies on CD26lowPD-1+ CD8 T cells from AML patients at initial diagnosis demonstrated that this cell population highly expressed inhibitory receptors and displayed impaired cytokine production, indicating an exhaustion status. Importantly, CD26lowPD-1+ CD8 T cells carried features of terminal exhaustion, manifested by higher frequency of TEMRA differentiation, increased expression of transcription factors that are observed in terminally exhausted T cells, and high level of intracellular expression of granzyme B and perforin. Our findings suggest a prognostic and predictive value of CD26 in AML, providing pivotal information to optimize the immunotherapy for this devastating cancer.