Optimizing CAR-T cell Culture: Differential effects of IL-2, IL-12, and IL-21 on CAR-T cells.

BACKGROUND: Chimeric antigen receptor (CAR)-T therapy has demonstrated sustained clinical remission in numerous hematologic malignancies and has expanded to encompass solid tumors and autoimmune diseases. While progress is being made in establishing optimal culture conditions for CAR-T cells, the identification of the most effective cytokine for promoting their persistence in vitro remains elusive. METHODS: Here, we employed scRNA-seq (single-cell RNA sequencing) analysis to investigate the potential alterations in biological processes within CAR-T cells following exposure to cytokines (IL-2, IL-12, and IL-21) and antigens. Transcriptomic changes in diverse CAR-T groups were compared following various treatments, with a focus on epigenetic modifications, metabolic shifts, cellular senescence, and exhaustion. RESULTS: Our study reveals that CAR-T cells treated with antigen, IL-2, and IL-12 exhibit signs of exhaustion and senescence, whereas those treated with IL-21 do not display these characteristics. The activities of glycolysis and epigenetic changes were significantly increased by treatments with antigens, IL-2, and IL-12, while IL-21 treatment maintained the oxidative phosphorylation (OXPHOS) of CAR-T cells. CONCLUSIONS: Our findings suggest that IL-21 may play a role in preventing senescence and could be utilized in combination with other strategies, such as IL-2 and IL-12, for CAR-T culture.

Multi-omics analysis reveals a feedback loop amplifying immune responses in acute graft-versus-host disease due to imbalanced gut microbiota and bile acid metabolism.

Acute graft-versus-host disease (aGVHD) is primarily driven by allogeneic donor T cells associated with an altered composition of the host gut microbiome and its metabolites. The severity of aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not solely determined by the host and donor characteristics; however, the underlying mechanisms remain unclear. Using single-cell RNA sequencing, we decoded the immune cell atlas of 12 patients who underwent allo-HSCT: six with aGVHD and six with non-aGVHD. We performed a fecal microbiota (16SrRNA sequencing) analysis to investigate the fecal bacterial composition of 82 patients: 30 with aGVHD and 52 with non-aGVHD. Fecal samples from these patients were analyzed for bile acid metabolism. Through multi-omic analysis, we identified a feedback loop involving "immune cell-gut microbes-bile acid metabolites" contributing to heightened immune responses in patients with aGVHD. The dysbiosis of the gut microbiota and disruption of bile acid metabolism contributed to an exaggerated interleukin-1 mediated immune response. Our findings suggest that resistin and defensins are crucial in mitigating against aGVHD. Therefore, a comprehensive multi-omic atlas incorporating immune cells, gut microbes, and bile acid metabolites was developed in this study and used to propose novel, non-immunosuppressive approaches to prevent aGVHD.

[Preliminary Study on the Effect of Silencing Nucleostemin Com- bined with Rapamycin on Autophagy and Apoptosis of HL-60 Cells].

OBJECTIVE: To investigate the effects of knocking down nucleostemin ( NS) combined with rapamycin (RAPA) on autophagy and apoptosis in HL-60 cells , and to explore its role in HL-60 cells . METHODS: The expression of NS protein was detected using Western blot , after transfection of HL-60 cells was achieved by the recombinant lentviral vector NS -RNAi-GV248 . Flow cytometry was used to detect changes in cells apoptosis after NS silencing/ rapamycin for 24 , 48 hours , and the expressions of NS , LC3 , p62 , BCL-2 and Bax proteins in cells were detected by Western blot. RESULTS: The expression of NS in HL-60 cells was successfully down-regulated by recombinant lentiviral vector. After treatment with rapamycin for 24 and 48 h , the apoptosis rate of cells in each group increased (P < 0.05) , and the apoptosis was more obvious at 48 hours . Compared with the NS silencing group or rapamycin group , after treated with NS down-regulation combined with rapamycin for 48 hours , the apoptosis of HL-60 cells was significantly increased ( P < 0.05 ) , LC3 -II/LC3 -I ratio was significantly increased ( P < 0.05 ) , p62 protein expression was significantly decreased (P < 0.05) , and BCL-2/Bax ratio was significantly decreased ( P < 0.05) . CONCLUSION: NS down-regulation combined with rapamycin can enhance the apoptosis and autophagy of HL-60 cells , and the induction of apoptosis of HL-60 cells may be related to the expression of BCL-2 and Bax proteins .

[Multiomics and Multidimensional Testing for Efficacy Monitoring of Patients with Lymphoma].

OBJECTIVE: To explore the role of a new blood-based, multiomics and multidimensional method for evaluating the efficacy of patients with lymphoma. METHODS: 10 ml peripheral blood was extracted from each patient, and the genomic copy number aberrations (CNA) and fragment size (FS) were evaluated by low-depth whole genome sequencing of cfDNA, and the level of a group of plasma tumor marker (PTM) were detected at the same time. The cancer efficacy score (CES) was obtained by standardized transformation of the value of above three numerical indexes, and the changes of CES before and after treatment were compared to evaluate the patient's response to the treatment regimen. RESULTS: A total of 35 patients' baseline data were collected, of which 23 cases (65.7%) had elevated CES values. 18 patients underwent the first time test. The results showed that the CES value of 9 patients with positive baseline CES decreased significantly at the first test, and the efficacy evaluation was PR, which was highly consistent with the imaging evaluation results of the same period. At the same time, the CNA variation spectrum of all patients were evaluated and it was found that 23 patients had partial amplification or deletion of chromosome fragments. The most common amplification site was 8q24.21, which contains important oncogenes such as MYC. The most common deletion sites were 1p36.32, 4q21.23, 6q21, 6q27, 14q32.33, and tumor suppressor-related genes such as PRDM1, ATG5, AIM1, FOXO3 and HACE1 were expressed in the above regions, so these deletions may be related to the occurrence and development of lymphoma. CONCLUSION: With the advantages of more convenience, sensitivity and non-invasive, this multiomics and multidimensional efficacy detection method can evaluate the tumor load of patients with lymphoma at the molecular level, and make more accurate efficacy evaluation, which is expected to serve the clinic better.

Combination of NeuX and NeuZ can predict neutrophil dysplasia features of myelodysplastic neoplasms in peripheral blood.

INTRODUCTION: The assessment of neutrophil dysplasia features in peripheral blood is very helpful for the early screening and diagnosis of myelodysplastic neoplasms (MDS). Cell population data (CPD) parameters generated by automated hematology analyzers can reflect morphological characteristics of blood cells. This study aimed to investigate the clinical significance of CPD parameters neutrophil (Neu) X, NeuY and NeuZ in assessing neutrophil dysplasia. METHODS: 218 MDS patients were divided into two subgroups according to neutrophil morphology. The differences of neutrophil research parameters between the two MDS subgroups and the control group, consisting of 210 healthy individuals, were compared, the correlation among neutrophil research parameters and the relationship between these parameters and cell morphology in MDS patients were analyzed, and receiver operating characteristic analysis were performed. RESULTS: The median values of neutrophil research parameters NeuX and NeuZ in MDS with granulocyte dysplasia group were significantly lower than those in MDS without granulocyte dysplasia group and control group (p < 0.001), and they were positively correlated (r = 0.878, p < 0.001). The area under the receiver operating characteristic curve of NeuX and NeuZ was 0.720 (95% CI: 0.643-0.796, p < 0.001) and 0.738 (95% CI: 0.665-0.811, p < 0.001), respectively. In addition, with the decrease of NeuX value, neutrophils gradually show decreased nuclear segment and/or cytoplasmic granules. CONCLUSIONS: Combining NeuX and NeuZ can predict neutrophil dysplasia features of MDS in peripheral blood, and this can be an easier method to screen for the neutrophil dysplasia cases, as compared with the microscopic examination of peripheral blood and/or bone marrow smears.

Differential diagnosis and identification of prognostic markers for peripheral T-cell lymphoma subtypes based on flow cytometry immunophenotype profiles.

We compared the differential expression of 15 markers in PTCL (Peripheral T-cell lymphoma) subtypes and T-CUS (T-cell clones of uncertain significance), and summarized the specific immunophenotype profiles of each subtype and its impact on prognosis. PD-1 and CD10 are diagnostic markers for AITL (angioimmunoblastic T-cell lymphoma). To avoid confusion with T-CUS of benign clones, it is recommended to define AITL as bounded by PD-1+%>38.01 and/or CD10+%>7.46. T cell-derived ENKTL-N (extranodal NKT cell lymphoma) specifically expresses CD56. ALCL (anaplastic large cell lymphoma) characteristically expresses CD30 and HLA-DR. PTCL-NOS (peripheral T-cell lymphoma unspecified) still lacks a relatively specific phenotype and is prone to loss of basic lineage markers CD3, CD5, and CD7. The determination of T-CUS can be verified by the overall assessment of the bone marrow and a certain period of follow-up. The clustering results showed that the expression of 8 specific markers was significantly different among the 5 groups, suggesting that a combination of related markers can be analyzed in the identification of PTCLs subtypes. The study explores the advantages of TRBC1 combined with CD45RA/CD45RO in detecting T cell clonality, which can efficiently and sensitively analyze multiple target T cell populations at the same time. The sensitivity of PB to replace BM to monitor the tumor burden or MRD (minimal residual disease) of PTCLs is as high as 85.71%, which can relieve the huge pressure of clinical sampling and improve patient compliance. CD7, CD38, and Ki-67 are prognostic indicators for AITL. CD3 and CD8 on PTCL-NOS, and CD56 and HLA-DR on ENKTL-N have prognostic role. This study supports and validates the current classification of PTCL subtypes and establishes an immunophenotypic profile that can be used for precise diagnosis. The important clinical value of PTCLs immunophenotype in routine classification diagnosis, clonality confirmation, prognosis prediction, and treatment target selection was emphasized.

The reference ranges and characteristics of lymphocyte parameters and the correlation between lymphocyte parameters and routine health indicators in adults from China.

BACKGROUND: Assessment of immune function is of key importance in recognition of disease or healthy status, which still faces challenge in clinical practice. We conducted a 10-center study to investigate lymphocyte parameters including the number, phenotype and IFN-γ-producing ability, and routine laboratory indicators by using the standard method. RESULTS: Although the heterogeneity of lymphocyte parameters was widely found, we have established the normal ranges of these parameters by using pooled data which showed no significant difference among centers. Cluster analysis of 35 parameters found 3 interesting clusters which represented different immunological status. Cluster 1 (parameters: IFN-γ+CD4+ T cell percentage and IFN-γ+CD8+ T cell percentage) represented current lymphocyte function, which was associated with indicators such as body mass index and red blood cell; Cluster 2 (parameters: NK cell number and CD45RA+CD4+ T cell percentage) represented potential of lymphocytes, which was associated with indicators such as albumin and high-density lipoprotein. Cluster 3 (parameters: HLA-DR+CD8+ T cell percentage) represented inflammatory status, which was associated with indicators such as low-density lipoprotein, globulin and age. Correlation analysis found that nutritional indicator albumin is significantly positively correlated with lymphocyte potential. Triglyceride and body mass index were positively correlated with current lymphocyte function rather than lymphocyte potential. The loss of CD8+ T cells was extremely pronounced with increasing age and was one of the most important factors to cause immunosenescence, which may be associated with increased glucose. CONCLUSIONS: We have established the normal ranges of lymphocyte parameters in different areas. This study elucidates the key indicators used to reflect the current function or potential of lymphocytes, which may provide a valuable clue for how to keep immunity healthy.

Comprehensive Analysis and Summary of the Value of Immunophenotypes of Mature NK Cell Tumors for Differential Diagnosis, Treatment, and Prognosis.

Background: Few studies have been performed to comprehensively analyze and summarize the immunophenotype and differential diagnosis of mature NK cell tumors, and there is often overlap between tumorigenic and reactive NK cell phenotypes. Furthermore, the impact of different phenotypes on patient prognosis has rarely been reported. Methods: The degree of expression of extracellular and intracellular markers of NK cells in each group was compared by FCM, and the differences in expression of various markers among different disease groups and their impact on prognosis have been analyzed and summarized. Results: Compared with normal NK cells, tumor cells of ANKL and ENKTL had characteristics of being more activated and progressive with larger FSC, in contrast to NK-CLPD and RNKL. Differential diagnoses with RNKL, ANKL, and ENKTL have broader FCM clues. In contrast, the phenotypes of NK-CLPD and RNKL are not significantly different, and consistent phenotypic abnormalities require ongoing monitoring to confirm malignant clones. The sensitivity of differentiating malignant NK cells from reactive NK cells by KIRs alone was poor. The clustering results showed that CD5, CD16, CD56, CD57, CD94, CD45RA, CD45RO, HLA-DR, KIRs, Granzyme B, Perforin and Ki-67 were differentially distributed in the expression of three NK cell tumors and reactive NK cell hyperplasia, so a comprehensive judgment using a wide range of antibody combinations is required in disease staging diagnosis. The tumor cell loads in BM and PB were also compared, and there was a clear correlation between the two. Moreover, the sensitivity of PB for monitoring tumor cells was up to 87.10%, suggesting that PB could be used as an alternative to BM for the diagnosis and screening of NK cell tumors. Analysis of the phenotypic impact of ENKTL patients on prognosis showed that those with CD7 and CD45RO expression had a poor prognosis, while those with positive KIRs had a better prognosis. Conclusion: This study systematically characterized the FCM of mature NK cell tumors, emphasizing the importance and clinical value of accurate immunophenotyping in diagnosing, classifying, determining prognosis, and guiding treatment of the disease.

Urinary neutrophil gelatinase-associated lipocalin rapidly decreases in the first week after kidney transplantation.

BACKGROUND: Recipient delayed graft function, which is defined as dialysis in the first week after transplantation, is one of the most common early complications after kidney transplantation. This study aimed to evaluate the daily changes in renal function-related biomarkers in the first week post-transplant. METHODS: A total of 72 kidney transplant recipients were retrospectively included in this study. Clinical and laboratory data were collected daily during the first week post-transplant, including urinary concentrations of neutrophil gelatinase-associated lipocalin (NGAL), serum concentrations of NGAL, creatinine, urea nitrogen, uric acid (UA), ß2-microglobulin, cystatin C, and estimated glomerular filtration rate (eGFR). RESULTS: There were no significant differences in urea nitrogen (P = .375), UA (P = .090), and cystatin C (P = .691), while urinary NGAL (P < .0001), serum NGAL (P < .0001), creatinine (P < .0001), ß2-microglobulin (P < .0001), and eGFR (P < .0001) were statistically significant in the first week post-transplant. In comparison with serum NGAL (P < .0001), creatinine (P < .0001), ß2-microglobulin (P = .001), and eGFR (P = .001), the change ratios of urinary NGAL changed the most between day 1 and day 2 after renal transplantation, while the changing degree of urinary NGAL showed no significant difference compared with these indicators between day 1 and day 7 after kidney transplantation. CONCLUSION: Urinary NGAL is a sensitive marker for indicating renal function. Urinary NGAL combined with other markers can be more helpful for evaluating renal function in the first week following kidney transplantation.

[Correlation of Nucleostemin with Programmed Death-ligand-1 in Multiple Myeloma U266 Cells].

OBJECTIVE: To investigate the correlation of nucleostemin (NS) gene with programmed death ligand-1 (PD-L1) in myeloma cells and the effect of NS expression down-regulation on the apoptosis of multiple myeloma cells, and to evaluate the associations among NS, PD-L1 and biological behavior of MM cells, and the feasibility of both NS and PD-1 as markers reflecting the status of MM cells. METHODS: The NS gene expression in U266 cells was down-regulated by NS-RNAi-GV248 recombinant lentivirus, the real-time PCR was used to detect the mRNA expression of NS, PD-L1 and PI3K/AKT/mTOR. The Western blot and flow cytometry were used to detect the expression of NS and PD-L1. The Annexin V-APC/7-AAD staining method was used to detect the apoptosis of U266 cells before and after knocking out the NS gene. RESULTS: Under the condition of MOI=10, the transfection efficiency was more than 75% by means of the fluorescent microscopy; real-time PCR showed that compared with the negative control group (1.002±0.026), the mRNA expression of NS, PD-L1 and PI3K/AKT/mTOR gene in the transfection group (0.415±0.089) was significantly reduced (P＜0.05). The results of flow cytometry and Western blot showed that the protein expression of PD-L1 was significantly down-regulated after transfection. After down-regulation of NS gene expression, the apoptosis of U266 cells increased (P＜0.05). CONCLUSION: The abnormal high expression of NS and PD-L1 genes exists in U266 cells, moreover, the down-regulation of PD-L1 and the related PISK/AKT/mTOR pathway gene expression appears after down-regulation of NS gene expression, which suggest that the cell biological changes resulted from above-mentioned results, show a synergestic effect on U266 cells.

Enrichment of circulating myeloma cells by immunomagnetic beads combined with flow cytometry for monitoring minimal residual disease and relapse in patients with multiple myeloma.

Difficulty in regularly analyzing marrow myeloma cells (MMCs) and low frequency of circulating myeloma cells (CMCs) in blood presents challenges for monitoring minimal residual disease (MRD) in multiple myeloma (MM). We have developed a set of method for enrichment of CMCs by immunomagetic beads (IMB) combined with flow cytometry (IMB-FCM) based on CD38-APC/CD138-APC antibodies in U266-spiked samples and in 122 patient samples. U266 cell capture efficiency of CD38/CD138-IMB-FCM (6.960, 2.574) was 6- and 2-fold higher than that of FCM (1.032), and the sensitivity of FCM and IMB-FCM was 0.01% and 0.001%, respectively. In MM cohort, the positive rate of CMCs by IMB-FCM increased from 60.5~70.0 to 85~87.2% in newly diagnosed/relapsed and partial remission (PR) patients compared with by FCM (P < 0.05). Two complete remission (CR) patients contain certain amounts of CMCs by IMB-FCM while no CMCs and MMCs were detectable by FCM. Patients exhibiting PR and CR upon therapy had much lower CMC and MMC counts than newly diagnosed/relapsed patients (P < 0.005). Based on MRD measurement in BM and PB samples, all FCM-negative BM samples were also paired with FCM/IMB-FCM-negative PB samples among newly diagnosed, relapsed, and PR patients, and FCM-positive BM samples were accompanied by IMB-FCM-positive results in 88% of corresponding PB samples. CMCs strongly associated with other clinical biomarkers of disease burden, including elevated MMCs, ß2-MG, sCrea, and DS and ISS stages, and more serious anemia, bone destruction, and renal impairment (P < 0.05). Logistic regression analysis revealed that elevated ß2-MG and moderate-to-more anemia were significant risk factors for the presence of CMCs (P < 0.05). As a noninvasive "liquid biopsy" of monitoring MRD, the potential of IMB-FCM for CMC detection may complement or minimize bone marrow aspiration in future treatment of MM patients.

Clinical characteristics and prognostic significance of EBER positivity in diffuse large B-cell lymphoma: A meta-analysis.

Recent studies show that Epstein-Barr virus (EBV) positivity might be related to adverse prognosis in patients with diffuse large B-cell lymphoma (DLBCL), but the results are still inconclusive. We conducted this meta-analysis to define the clinical value of EBV infection in DLBCL. All potential articles in PubMed, Web of Science, Medline, and Embase were retrieved. Using the random-effects or fixed-effect model, pooled hazard ratios (HRs) or relative risk (RR) with 95% confidence intervals (CIs) were used to calculate the correlation between EBER and prognosis and clinical features in DLBCL. A total of 13 qualified studies with 4111 patients were identified in our meta-analysis based on the inclusion and exclusion criteria. The overall estimates revealed that EBV-encoded small RNAs (EBER) positivity was significantly correlated with worse overall survival (HR = 2.43, 95% CI: 1.73-3.36) and progression-free survival (HR = 3.60, 95% CI: 2.07-6.26). In addition, EBER positivity was associated with age older than 60 years (RR = 1.51, 95% CI: 1.02-2.24), male sex (RR = 1.34, 95% CI: 1.05-1.71), more advanced stage (RR = 2.25, 95% CI: 1.72-2.96), high international prognostic index (RR = 2.20, 95% CI: 1.71-2.82), more than one extranodal involvement (RR = 1.69, 95% CI: 1.27-2.26), presence of B symptom (RR = 1.75, 95% CI: 1.30-2.35), non-germinal center B-cell subtype (RR = 1.35, 95% CI: 1.03-1.78), and elevated lactate dehydrogenase levels (RR = 1.30, 95% CI: 0.98-1.72). EBER positivity was correlated with worse outcomes, worse clinical course, and adverse clinicopathologic features among patients with DLBCL.

[Influence of Nucleostemin Expression Down-Regulation on PI3K/AKT/mTOR Signal Pathway in HL-60 Cells].

OBJECTIVE: To explore the effect of nucleostemin(NS) RNAi on the expression of signal molecules in PI3K/AKT/mTOR pathway, a candidate of p53-independent signal pathway in the leukemia HL-60 cells. METHODS: The expression of NS was interfered by transfection of P53-deficient HL-60 cells with the recombinant lentivirus expression vector NS-RNAi-GV248. The exression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by Western blot. RESULTS: The fluorescence microscopy showed that the recombinant lentivirus vector NS-RNAi-GV248 transfected HL-60 cells successfully with a 80% transfection rate. Western blot showed that the expression of NS protein was inhibited obviously in HL-60 cells, and the expression levels of AKT, p-AKT, p70s6k and p-p70s6k were not statistically different(t1=2.31,P>0.05;t2=3.62,P>0.05;t3=1.60,P>0.05;t4=2.72,P>0.05) in comparison with control; the expression of GßL protein was statistically down-regnlated (t=15.01,P=0.002). CONCLUSION: The changes of GßL protein correlats with NS knockdown. The PI3K/AKT/mTOR pathway may be one of nucleostemin p53-independent signal pathways.

Prognostic value of circulating plasma cells in patients with multiple myeloma: A meta-analysis.

PURPOSE: The clinical significance and prognostic role of circulating plasma cells (CPCs) in multiple myeloma (MM) are still controversial. We conducted the first meta-analysis to clarify the correlation between CPCs and the clinicopathological features and prognosis of MM patients. METHODS: A comprehensive literary search for relevant studies was performed on PubMed, Embase, Medline, CNKI (Chinese) and Web of Science databases (January 1, 1950 to December 20, 2016). The associations between CPCs and survival rate and clinicopathological parameters, including International staging system (ISS) and Durie-Salm staging system (DS) stage, were evaluated. Then pooled hazard ratios (HRs) for survival with 95% confidence intervals (CIs), subgroup analysis, sensitivity analysis, and publication bias were conducted. RESULTS: 11 studies covering a total of 2943 patients were included. Pooled hazard ratios (HRs) revealed that the presence of CPCs predicted aggressive disease progression (HR = 1.78, 95% CI = 1.57-2.03) and reduced overall survival (OS) (HR = 1.82, 95% CI = 1.59-2.08). Subgroup analyses demonstrated that CPCs positive patients also had poor disease progression and OS in detection methods and sampling time subsets. Moreover, the presence of CPCs was strikingly associated with increased ISS stage (OR = 2.78% CI = 1.69-4.56), but not with DS stage(OR = 1.60; 95% CI = 0.74-3.47). CONCLUSIONS: CPCs status is associated with poorer survival outcome in multiple myeloma. Additionally, increased ISS stage could be significant risk factors for the presence of CPCs.

[Effect of Down-Regulatory Nucleostemin Expression on Autophagy Activities in p53 Null HL-60 Leukemia Cells].

OBJECTIVE: Based on previous microarry and bioinformatic analysis results, to investigate the effect of nucleostemin(NS) expression down-regulation on autophagy activity in p53 null HL-60 leukemia cells, so as to provide evidence for studying mechanisms of p53-independent signal pathway of NS in details. METHODS: The autophagy activity of HL-60 cells after down-regulation of NS expression was detected with acidine orange staining, Western blot and transmission electron mcrioscope technique. RESULTS: The expression level of NS in test groups was lower than that in blank control and negative control groups after HL-60 cells were readily transinfected by lentivirus. The result of acidine orange staining showed that the number of acid vesicular organelle in test groups(22.4±0.76)% was higher than that in blank control groups(3.1±0.28)% and negative control groups(6.2±0.64)% (P<0.05). Western blot showed that the ratio of LC3II/LC3I in test groups(1.537±0.072) was higher than that in blank control and negative control groups (1.010±0.039) and (0.608±0.008). The result of transmission electron mcrioscopy also showed that the number of autophagosomes in test group(8.7±3.1) was higher than that in the blank control and negative control groups(4.2±1.2) and (2.3±0.5). CONCLUSION: Autophagy activty can be enhanced after the level of NS was down regulated. The change indicates the signaling transductions screened by bioinformatic analysis may be one of p53-independent pathway of NS, which lays a foundation for contineously studying key points of p53-independent signal pathway of NS.

[Expression of CC-chemokine Receptor 7 in Patiens with Multiple Myeloma and Its Relationship with Extramedullary Disease].

OBJECTIVE: To investigate the expression of CC-chemokine receptor 7(CCR7) in patients with multiple myeloma(MM) and its correlation with clinical features of MM. METHODS: The level of CCR7 expression in bone marrow samples from 53 newly diagnosed MM patients was detected by flow cytometry(FCM). Statistical methods were used to analyze the correlation between CCR7 expression and clinical features, such as sex, age, M protein, peripheral blood cell count, biochemical indicators, plasma cell ratio of bone marrow, immunophenotype, osteopathy and extramedullary disease. RESULTS: The plasma cells in 24 out of 53 cases(45.28%) expressed CCR7. The rate of extramedullary disease in CCR7 positive group was significantly higher than that in CCR7 negative group (29.17% vs 3.45%)(P<0.05). CONCLUSION: The expression of CCR7 in patients with MM is high, moreover this high expression correlates with extramedullary disease, thus CCR7 can be used as an effective indicator for prediction of extramedullary disease.

Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays.

Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53­mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53­independent NS pathway. NS expression was silenced using lentivirus­mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse­transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c­Jun N­terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54­independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia.

[Expression and Clinical Significance of CC-chemokine Receptor 7 in Adult Acute Leukemia].

OBJECTIVE: To explore the expression of CC-chemokine Receptor 7 (CCR7) in adult acute leukemia patients, and to analyze the relationship of CCR7 expression with the clinical characteristics of patients. METHODS: The expression of CCR7 in bone marrow samples from adult acute leukemia patients were detected by flow cytometry (FCM), the relationship of CCR7 expression with the clinical characteristics of patients such as sex, age, WBC count, blast cell ratio, CD56 expression, molecular biology, cell genetics, risk stratification, extramedullary infiltration was analyzed. RESULTS: The expression rate of CCR7 in adult ALL and AML patients was 36.8% and 9.6%, respectively, and the expression level of CCR7 in ALL patients was higher than that in AML patients (P < 0.05). The extramedullary infiltration rate was 100% and 41.7 % for CCR7 positive and negative groups of ALL, respectively (P < 0.05). While the mean fluorescence intensity (MFI) in extramedullary infiltration group of ALL was higher than that in none-extramedullary infiltration group of ALL (50.00 ± 10.42 vs 18.14 ± 1.39), respectively (P < 0.05). CONCLUSION: CCR7 is higher expressed in adult acute leukemia cells, moreover its expression rate in ALL is higher than that in AML, and the expression of CCR7 is related with extramedullary infiltration in ALL.

[Effect of Dexamethasone on Blast Composition in Patients with Myelodysplastic Syndrome and Its Diagnostic Significance].

OBJECTIVE: To analyze the effect of dexamethason (Dex) on blast composition in patients with myelodysplastic syndrome (MDS) and investigate its significance in diagnosis of MDS. METHODS: The flow cytometry (FCM) was used to detect the blast rate and the expression of its antigens in 30 cases of MDS (10 cases were treated with Dex as DX group and 20 cases were treated without Dex as control group). RESULTS: The difference of the CD34(+) cell number detected by FCM was not statistically significant between DX group and control group (P > 0.05); The rate of BM B cell precursors (BCP CD34(+)/CD19(+)/CD10(+) cells) increased in DX group significantly, and BM CD117(+) cells in CD34(+) cells was decreased significantly as compared with control group (P < 0.001). The expression of antigens between granulocyte and monocyte was not significantly different (P > 0.05). CONCLUSION: The dexamethasone can increase the rate of BCP significantly and decreased the rate of BM CD117(+) cells in CD34(+) cells significantly. There is significant influence on the blast composition in MDS patients after dexamethasone treatment and without significant influence on the other phenotypcs.

[Expression of CD58 in childhood B-lineage acute lymphoblastic leukemia and its feasibility in minimal residual disease detection].

OBJECTIVE: To measure the expression of lymphocyte function-associated antigen-3 (CD58) in childhood B-lineage acute lymphoblastic leukemia (B-ALL) and to explore the feasibility of CD58 as an indicator for minimal residual disease (MRD) detection in childhood B-ALL. METHODS: Eighty-seven children diagnosed with B-ALL between January 2014 and September 2014 were enrolled, and 20 hospitalized children who had no tumor or blood disease and had normal bone marrow cell morphology served as the control group. The expression features of CD58 in bone marrow samples from the two groups (at diagnosis, on day 15 of induction chemotherapy) were analyzed by four-color flow cytometry (FCM). Quantitative real-time polymerase chain reaction (qRT-PCR) and FCM were used to detect MRD in B-ALL patients on day 33 of induction chemotherapy. RESULTS: The mean fluorescence intensity of CD58 expression in the 87 B-ALL cases (91±33) was significantly higher than that in the 20 controls (14±6) (P<0.01); CD58 was over-expressed in 44 of the B-ALL cases. In the B-ALL children, the expression of CD58 on day 15 of induction chemotherapy (105±22) was not significantly different from that at diagnosis (107±26) (P>0.05). In the 44 B-ALL patients with CD58 over-expression, FCM showed 9 MRD(+) cases and 35 MRD(-) cases, while qRT-PCR showed 11 MRD(+) cases and 33 MRD(-) cases; 42 cases (95%) showed consistent results of the two tests, so there was no significant difference between the two methods in detecting MRD (P>0.05). CONCLUSIONS: CD58 is over-expressed and stable in children with B-ALL, and it can be considered as an indicator for MRD detection in childhood B-ALL.