[The Prognostic Value of Prognostic Nutritional Index Combined with D-dimer in Patients with Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To explore the effects of prognostic nutritional index (PNI) combined with D-dimer on the prognosis of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL). METHODS: The clinical data of 73 DLBCL patients at initial diagnosis were retrospectively evaluated, and the optimal cut-off point of PNI and D-dimer were determined by ROC curve. The overall survival (OS) rate and progression-free survival (PFS) rate in different subgroups were compared using Kaplan-Meier survival curves. Univariate and multivariate Cox regression analysis was performed to identify the factors associated with OS. RESULTS: Compared with the low PNI group (PNI<44.775), the high PNI group (PNI≥44.775) had better OS (P =0.022) and PFS (P =0.029), the 2-year OS rates of the two groups were 55.6% and 78.3% respectively (P =0.041). Compared with the high D-dimer group (D-dimer≥0.835), the low D-dimer group (D-dimer<0.835) had better OS (P <0.001) and PFS (P <0.001), the 2-year OS rates of the two groups were 51.4% and 86.8% respectively (P =0.001). Meanwhile, patients in the high PNI+ low D-dimer group had better OS (P =0.003) and PFS (P <0.001) than the other three groups, the 2-year OS rate was statistically different from the other three groups (P <0.05). The multivariate analysis revealed that NCCN-IPI (HR =2.083, 95%CI : 1.034-4.196, P =0.040), PNI (HR =0.267, 95%CI : 0.076-0.940, P =0.040) and PNI+D-dimer (HR =9.082, 95%CI : 1.329-62.079, P =0.024) were the independent risk factors affecting OS in patients with DLBCL. Subgroup analysis showed that PNI, D-dimer, and PNI combined with D-dimer could improve the prognostic stratification in low and low-intermediate risk DLBCL patients. CONCLUSION: High PNI, low D-dimer and combination of high PNI and low D-dimer at initial diagnosis suggest a better prognosis in DLBCL patients.

[Prognostic Value of A New Risk Stratification Model (R2-ISS) in Newly Diagnosed Multiple Myeloma].

OBJECTIVE: To investigate the significance of a new risk stratification model (R2-ISS) in evaluating the prognosis of newly diagnosed multiple myeloma (MM). METHODS: Clinical data of 116 newly diagnosed MM patients admitted to Lanzhou University Second Hospital from June 2012 to March 2021 were retrospectively analyzed. According to R2-ISS, these patients were divided into four groups: low risk, low-intermediate risk, intermediate-high risk, and high risk. The significance of R2-ISS on prognosis of the patients was analyzed. RESULTS: Survival analysis showed that R2-ISS was associated with progression-free survival (PFS) (P=0.042) and overall survival (OS) (P=0.014). Cox univariate analysis showed that lactate dehydrogenase, serum calcium, serum creatinine, ß2-microglobulin, ISS, R-ISS, R2-ISS, t(4;14), and autologous hematopoietic stem cell transplantation (ASCT) were the influencing factors of OS in newly diagnosed MM patients (all P<0.05). Cox multivariate analysis showed that R-ISS, R2-ISS, and ASCT were independent risk factors affecting OS (all P<0.05). In addition, survival analysis of patients with different R2-ISS showed that ASCT improved PFS and OS. CONCLUSION: R2-ISS has prognostic value for newly diagnosed MM patients, while ASCT can improve the prognosis of patients with different R2-ISS.

[A Prognostic Nomogram Based on Response to Bortezomib and BTK Expression for Treatment-Experienced Multiple Myeloma Patients].

OBJECTIVE: To establish a prognostic nomogram based on response to bortezomib and BTK expression for treatment-experienced multiple myeloma patients. METHODS: The Oncomine database was utilized to determine BTK expression, sex, age, albumin, Mayo index, response to bortezomib treatment, follow-up time and survival status in multiple myeloma(MM) patients. Cut-off point for BTK expression was calculated using R software. Univariate and multivariate analyses by Cox proportional hazards regression were then performed. Significant prognostic factors were combined to build a nomogram. The discrimination ability and predictive accuracy of the nomogram were evaluated using the index of concordance (C-index) and calibration curves. RESULTS: Multivariate analysis showed that response to bortezomib, BTK expression and sex were independent risk factors for prognosis. The C-index value of the nomogram made according to the independent risk factors was 0.729 (95%CI, 0.642-0.8164). The calibration curves showed good consistency between predicted and actual survivals for 1-year and 2-year overall survival. CONCLUSION: The proposed nomogram is accurate in predicting the prognosis of patients with MM.

[The Influence of CONUT Score on the Prognosis of Patients with Multiple Myeloma].

OBJECTIVE: To explore the influence of controlling nutritional status (CONUT) score on the prognosis of newly diagnosed patients with multiple myeloma (MM). METHODS: The clinical data 119 patients with MM who were diagnosed according to the international myeloma diagnostic criteria in Lanzhou University Second Hospital from April 2010 to October 2018 were collected and retrospectively analyzed. The relationship between clinical indexes, including age, sex, MM type, absolute lymphocyte count (ALC), absolute neutrophil count (ANC), absolute monocyte count (AMC), hemoglobin (Hb), platelet (PLT), ß2-microglobulin (ß2-MG), lactate dehydrogenase (LDH), albumin (ALB), globulin (GLO), cholesterol (CHO), serum creatinine (Scr), etc, and CONUT score was discussed to explore the prognostic value of these indicators. SPSS 25.0 software was used for statistical analysis. Progression-free survival(PFS) and overall survival (OS) between different subgroups were calculated by Kaplan-Meier curves and difference between survival curves was detected by Log-rank tests. Receiver operating characteristic (ROC) curve was used to estimate the most discriminative cutoff value of CONUT score for predicting OS. Mann-Whitney U test was used for non-parametric samples, and chi-square test for categorical variables. Univariate and multivariate analysis were performed by using COX proportional hazards model to identify factors associated with OS. RESULTS: Compared with high-scoring group, low-scoring group had a better OS ï¼»median OS was 43.3 months and 127.67 months, respectively, 95% confidence interval (CI): 57.065-78.345, P=0.038ï¼½. At the same time, the low-scoring group also had higher level of ALC, ANC, AMC, Hb, PLT, ALB, and CHO but lower of GLO. Multivariate survival analysis showed that age (HR=1.027, 95%CI: 1.000-1.054, P=0.048), AMC (HR=11.284, 95%CI: 22.968-42.897, P<0.001), CONUT score (HR=1.198, 95%CI: 1.036-1.385, P=0.015), M protein (non-IgG/IgG type) type (HR=0.503, 95%CI: 0.259-0.977, P=0.043) were independent factors affecting the prognosis of MM patients. CONCLUSION: The CONUT score as an immune-nutrition score is a convenient and easy-to-obtain index to effectively predict the prognosis of MM patients.

[Value of Red Blood Cell Distribution Width and Fibrinogen Level for Evaluation of the Therapeutic Efficacy and Prognosis in Patients with Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To investigate the value of red blood cell distribution width (RDW) and fibrinogen (Fib) level for the evaluation of therapeutic efficacy and prognosis in patients with diffuse large B-cell lymphoma (DLBCL). METHODS: The relationship between RDW/Fib at initial diagnosis and efficacy and the clinical outcome was retro-spectively analyzed based on the study of 105 patients with DLBCL. The patients were divided into two groups: low RDW group (≤15%) and high RDW group (>15%), low Fib group (Fib≤4 g/L) and high Fib group (Fib>4 g/L) according to the normal values of RDW and Fib. Therapeutic efficacy, overall survival (OS) time and progression free survival (PFS) time were compared between two groups. The correlation between each factors and efficacy, prognosis was analyzed by univariate and multivariate regression. RESULTS: The therapeutic efficacy (P<0.001), OS time(P=0.004), and PFS time(P=0.007) were poorer in the high RDW group as compared with the low RDW group. The efficacy (P=0.015) and PFS time(P=0.04) were poorer in the high Fib group as compared with the low Fib group. Multivariate analysis showed that high RDW was the independent risk factor for efficacy of DLBCL patients (OR=3.394, 95% CI 1.093-10.539, P=0.035). CONCLUSION: High RDW and high Fib associate with poor efficacy in DLBCL patients.

[Effect of plasmacytoid dendritic cells activited by bacteria on spontaneous remission of leukemia].

Spontaneous remission (SR) of leukemia is a rare event in clinic, which possibly correlated with severe infection and sepsis, but its exact mechanism has not been confirmed. Plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) play a key role in innate and adaptive immunity respectively. A patient with severe infection of staphylococcus aureus acquired completely spontaneous remission (SR), moreover a increased number of pDC were observed, suggesting that bacteria-activated pDC may play an important role in SR. This study was purposed to explore if the bacteria can stimulate pDC successfully and get a functional pDC. Both pDC and mDC were isolated from freshly collected, leukocyte-rich buffy coats from healthy blood donor and leukemic patient with SR by using MACS and FACS. The pDC were cultured in RPMI 1640 medium and were stimulated with different kinds of bacteria and the expression of CD40, CD86 and HLA-DR on the cell surface was analyzed by flow cytometry. The cytokine (IFN-α, IL-12, IFN-γ, IL-2, IL-4, IL-10) production was measured by using ELISA kits. The results showed that the stimulation with staphylococcus aureus and pseudomonas aeruginosa resulted in the maturation of pDC, which secrete a large number of IFN-α and promote the differentiation of naive CD4⁺ T cells to Th1 cells. The activated pDC expressed high level of CD40 and CD86 and showed higher T cell stimulatory capacities. It is concluded that staphylococcus aureus and pseudomonas aeruginosa can activate pDC, the activated pDC secrete high quantity of IFN-α. This result suggests that bacteria stimulated pDC may play a key role in SR of leukemia following severe infections.

[Effect of astragalus polysaccharide on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism].

The objective of this study was to explore the effect of astragalus polysaccharide (APS) on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism. The cytotoxicities of NK cells against HL-60 cells were analyzed by LDH releasing assay at different effect-to-target cell ratios (E:T) before and after treated with APS. The gene expression of MHC class I chain-related (MICA) in HL-60 cells before and after APS treatment was assayed with RT-PCR. Protein expression of MICA in HL-60 cells was assayed by flow cytometry before and after treated by APS. The results showed that after treated with APS 15 mg/ml for 48 h, the cytotoxicities of NK cells against HL-60 cells enhanced at different effect-to-target (P < 0.05), and the gene and protein expressions in MICA of HL-60 cells were up-regulated (P < 0.05). It is concluded that the APS can obviously up-regulate the expression of MICA in HL-60 cells, thus enhance sensitivity of HL-60 cells to cytotoxicity of NK cells.

[Effect of high dose dexamethasone on function and TLR-9 mRNA expression of plasmacytoid dendritic cells in patients with immune thrombocytopenic purpura].

This study was purposed to investigate the effect of high-dose dexamethasone (DXM) on function and Toll like receptor 9 (TLR-9) expression of plasmacytoid dendritic cells (pDC) in peripheral blood of patients with immune thrombocytopenic purpura (ITP). 15 newly diagnosed patients with ITP received high dose DXM at single daily doses of 40 mg for 4 consecutive days. The peripheral blood plasmacytoid dendritic cells from 13 remission patients and 15 normal controls were separated by immunomagnetic beads and then induced by CpG-OND2216. 24 h later, the levels of IFN-α, IL-6 and TNF-α in the supernatant were detected by enzyme linked immunosorbent assay (ELISA). The expression of TLR9 mRNA of pDC was detected by real-time quantitative PCR. The results indicated that the levels of IFN-α, IL-6 and TNF-α produced by pDC in ITP patients were significantly higher than those in normal controls (P < 0.05). After high dose DXM treatment, the levels of IFN-α, IL-6 and TNF-α decreased without significant difference compared with normal controls (P > 0.05). The expression of TLR9 mRNA in pDC of untreated patients was significantly higher than that in control group (P < 0.05), and significantly reduced after treatment without difference from that in control group (P > 0.05). It is concluded that pDC may play an important role in ITP by their TLR9 and secreted cytokines; dexamethasone may down regulate the expression of TLR9, inhibit pDC function, and thus play a therapeutic role.

[The immune effects of rituximab on dendritic cells derived from patients with primary immune thrombocytopenia].

OBJECTIVE: To explore the changes of surface antigen and function of rituximab on dendritic cells derived from patients with Primary immune thrombocytopenia (ITP) to further understand the effective mechanism of immunotherapy. METHODS: The peripheral blood mononuclear cells (PBMCs) were isolated from remission patients with ITP before and after low-dose rituximab infusion, and the PMNCs were stimulated for 5 days by rhGM-CSF and rhlL-4 in 5% CO2 air at 37°C incubator. Then all of DCs were cultured with TNF-α for 48 hours. The morphology of DCs was monitored under inverted microscope daily, and the surface antigens of the DCs were analysed by flow cytometry, meanwhile the levels of IL-12p70 and TGF-ß1 in supernatants were detected by ELISA, mix lymphocyte reaction was performed by MTT assay. RESULTS: (1) Rituximab-treated-DCs showed no obvious tree-like protruding compared with untreated-DCs. The former cells were small and most of nucleus were centric. (2) The expressions of HLA-DR, CD80, CD83 and CD86 on rituximab-treated-DCs \[56.37 ± 3.95)%, (36.41 ± 2.82)%, (30.45 ± 4.61)% and (41.98 ± 4.17)%, respectively\] were significantly lower than those untreated-DCs \[(73.71 ± 7.61)%, (55.14 ± 7.30)%, (80.91 ± 7.09)% and (59.03 ± 3.43)%, respectively\](all P < 0.05), the concentration of IL-12p70 was significantly lower, \[(66.87 ± 4.29)% vs (50.17 ± 14.52)%\], while that of TGF-ß1 \[(9.70 ± 0.31)%\] higher than the untreated-DCs \[(2.70 ± 0.36)%\] (P < 0.05). (3) The abilities to activate T cells proliferation of rituximab-treated-DCs reduced compared with untreated-DCs. CONCLUSION: The surface antigen of ITP-DCs and the concentration of IL-12p70 reduced after the low-dose rituximab infusion. The abilities to activate T cells proliferation reduced while the concentration of TGF-ß1 increased. Rituximab may achieve its therapeutic effect on ITP by downregulating the immunoreactivity of DCs.

[Anti-tumor immune response of dendritic cells derived from lymphoma cells transduced with recombinant adenovirus encoding human P53].

This study was aimed to investigate the immunological effect of modified dendritic cells (DC) which inducing cytotoxic T cells (CTL) against lymphoma cells. The DC were isolated from the lymph node and peripheral blood of patients with diffuse large B cell lymphoma (DLBCL). DC were transfected with recombinant adenovirus vector carrying human p53 gene (rAd-p53-DC). The expression of p53 gene was detected by flow cytometry. Western-blot was used to detect the expression of P53. ELISA was used to detect IL-12 level in supernatant. The mixed lymphocyte reaction (MLR) was used to detect the proliferative ability of auto-lymphocyte stimulated by DC. The lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTL. The results indicates that the expressions of DC surface molecule (except for CD1a) such as CD83, CD80, CD86 and HLA-DR were significantly higher in experiment group than that in control group and blank control group. The secretion of IL-12 in supernatant was higher in experiment group than that in control group. The autologous T lymphocyte proliferation and cytotoxic activity against the same kind of DLBL-cells increased in experiment group as compared with control group and blank control group (P < 0.05). The ability to stimulate T lymphocyte proliferation increased with the rising of the ratio of DC and T lymphocyte. However, there was statistically significant difference between rAd-p53-DC derived from Lymph node and peripheral blood (P < 0.05). It is concluded that rAd-p53-transfected DC can induce CTL response in vitro against lymphoma cells.

[The prevalence of Th17 cells in patients with acute myeloid leukemia].

OBJECTIVE: To investigate the proportion of Th17 cells in the peripheral blood of the patients with acute myeloid leukemia (AML) and evaluate the potential association of Th17 cells with AML. METHODS: The cytokines IL-17 and TGF-ß1 in the peripheral blood of AML patients before therapy (group 1), AML patients in complete remission (AML-CR, group 2) and healthy donors (group 3) were measured by enzyme-linked immunosorbent assay (ELISA). The proportion of Th17 cells of each group was evaluated by flow cytometry. The level of IL-17 mRNA of each group was examined by reverse transcription-PCR (RT-PCR). RESULTS: The percentage of Th17 cells and the level of IL-17, IL-17 mRNA in group 1 \[(10.502 ± 1.071) ng/L, (0.935 ± 0.140)% and 0.262 ± 0.510\] and group 2 \[(11.345 ± 0.987) ng/L, (1.091 ± 0.159)% and 0.307 ± 0.031\] was significantly lower than that in group 3 \[(16.852 ± 1.198) ng/L, (2.586 ± 0.235)% and 0.501 ± 0.060\]. The percentage of Th17 cells and the level of IL-17, IL-17 mRNA in group 1 was lower than that in the group 2. But the level of TGF-ß1 in the group 1 (29.963 ± 1.588) ng/L and the group 2 (25.163 ± 1.848) ng/L was significantly higher than that in group 3 (13.366 ± 1.565) ng/L. However, the level of TGF-ß1 in the group 3 was higher than that of the group 2. CONCLUSION: Th17 cells might be negatively correlated with the AML development. The overexpression of TGF-ß1 in AML patients might suppress the differentiation of Th17 cells.

[Killing activity of co-cultured cytokine-induced killer cells and dendritic cells against multi-drug resistant tumor cell lines].

OBJECTIVE: A lot of studies have suggested that a certain amount of T cells may be involved among cytokine-induced killer (CIK) cells. The aim of the present study was to prove whether an antigen-specific killing effect on tumor cells is involved during the CIKs-induced killing process. METHODS: Bone marrow mononuclear cells (BMMNCs) derived from healthy subjects were separately cultured to generate dendritic cells (DC) and CIKs. A human mammary cancer cell line MCF-7/ADR, expressing P-gp antigen, was frozen-thawed and the lysate including P-gp antigen was obtained. The DC pulsed with or without tumor antigen lysate was co-cultured with CIK (pulsed-DC + CIK and DC + CIK), and CIK cultured alone was used as control. The cell phenotype of DC and CIK was analyzed by flow cytometry. The secretion of IL-12 and IFN-gamma was assayed by ELSA. The antitumor effect of the three CIK groups targeted at MCF-7/ADR cells expressing P-gp antigen and MCF-7 cells was detected by MTT. RESULTS: Pulsed-DC + CIK group and DC + CIK group showed a higher expression level of DC mature phenotypes than those before co-culture with CIK, with a significant difference (P = 0.003, P = 0.001, respectively). The phenotypes (CD3, CD8, CD56) of CIK in pulsed-DC + CIK group and DC + CIK group was higher than those in CIK group (P = 0.003, P = 0.011, respectively). Among the three CIK groups, pulsed-DC + CIK group had the highest phenotypes on CD3+ CD56 (pulsed-DC + CIK vs. DC + CIK, P = 0.001; pulsed-DC + CIK vs. CIK, P < 0.001) and CD3 CD8 (P = 0.002, P = 0.002, respectively). Among the three groups, the pulsed-DC + CIK group showed the lowest CD45RA phenotype (pulsed-DC + CIK vs. DC + CIK, P < 0.001; pulsed-DC + CIK vs. CIK, P = 0.004). Among the three groups the secretion of IL-12 and IFN-gamma had the highest level in pulsed-DC + CIK group, with a value of 254 +/- 14.5 pg/ml and 3100 +/- 286 pg/ml, respectively. The antitumor killing effect on MCF-7/ADR cells had a significant difference between any two groups (pulsed-DC + CIK VS. DC + CIK, P = 0.039; pulsed-DC + CIK VS. CIK, P = 0.002; DC + CIK vs. CIK, P = 0.049). The highest was in pulsed-DC + CIK group and the lowest was in CIK group. The CIK group showed a significantly lower antitumor effect on MCF-7 cells than the other two groups (pulsed-DC + CIK vs. CIK, P = 0.007; DC + CIK vs. CIK, P = 0.048), but no significant difference between the pulsed-DC + CIK and DC + CIK groups. CONCLUSION: In the present study, DC and CIK cells have been successfully obtained and cultured from bone marrow mononuclear cells. After their co-culture, not only both their specific phenotypes were increased, but also the associated cytokines were secreted. An improved antitumor killing effect and some possible specific immunocytotoxicity were observed. Our findings provided a basis for experimental and clinical research on bio-immunotherapy targeted at multi-drug resistant tumor cells.

[Induction of anti-leukemic immunity of dendritic cells derived from multidrug resistant leukemia K562/A02 cells with high expression of P-glycoprotein and sensitive K562 cells].

This study was aimed to investigate and compare the anti-leukemic effect mediated by dendritic cells (DC) derived from multidrug resistant (MDR) leukemia K562/A02 cells with high expression of p-glycoprotein (P-gp) and sensitive K562 cells. Multidrug resistant K562/A02 cell line and sensitive K562 cell line from chronic myeloid leukemia (CML) were induced for differentiating to DC in complete RPMI 1640 culture medium supplemented with GM-CSF (1 000 U/ml), IL-4 (500 U/ml) and TNF-alpha (100 ng/ml) for 14 days. The morphologic features of DC were observed by means of optical microscopy and the phenotype of DC was detected by flow cytometry. T-cell stimulating activity was determined by allogeneic lymphocyte reaction (allo-MLR). Cytotoxic activity was measured by MTT assay. The results indicated that DC derived from K562/A02 cells and K562 cells similarly showed the typical morphology of dendritic cell and expressed the surface differentiation antigens and costimulatory molecules CD1a, CD83, HLA-DR, CD80 and CD86 of DC. In allo-MLR, K562/A02-DC had a higher capacity to induce lymphocyte proliferation, compared with K562-DC (P < 0.05). K562/A02-DC and K562-DC could similarly generate specific cytotoxic activity against K562/A02 cells or K562 cells respectively, but low reactivity against HL-60 cells. More importantly, the cytotoxic activity mediated by K562/A02-DC was stronger than that by K562-DC against K562/A02 cells or HL-60/VCR cells (P < 0.01, respectively). It is concluded that functional DC can be differentiated from multidrug resistant leukemia K562/A02 cells as well as sensitive K562 cells in the presence of GM-CSF, IL-4 and TNF-alpha. Especially, DC derived from K562/A02 cells can induced a p-glycoprotein specific anti-leukemic immunity.