Multiomics and Multidimensional Testing for Efficacy Monitoring of Patients with Lymphoma / 中国实验血液学杂志

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.

Correlation of Nucleostemin with Programmed Death-ligand-1 in Multiple Myeloma U266 Cells / 中国实验血液学杂志

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.

Effect of Down-Regulatory Nucleostemin Expression on Autophagy Activities in p53 Null HL-60 Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Influence of Nucleostemin Expression Down-Regulation on PI3K/AKT/mTOR Signal Pathway in HL-60 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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(t=2.31,P>0.05;t=3.62,P>0.05;t=1.60,P>0.05;t=2.72,P>0.05) in comparison with control; the expression of GβL protein was statistically down-regnlated (t=15.01,P=0.002).</p><p><b>CONCLUSION</b>The changes of GβL protein correlats with NS knockdown. The PI3K/AKT/mTOR pathway may be one of nucleostemin p53-independent signal pathways.</p>

Expression of CC-chemokine Receptor 7 in Patiens with Multiple Myeloma and Its Relationship with Extramedullary Disease / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of CC-chemokine receptor 7(CCR7) in patients with multiple myeloma(MM) and its correlation with clinical features of MM.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Expression and Clinical Significance of CC-chemokine Receptor 7 in Adult Acute Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Effect of Dexamethasone on Blast Composition in Patients with Myelodysplastic Syndrome and Its Diagnostic Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the effect of dexamethason (Dex) on blast composition in patients with myelodysplastic syndrome (MDS) and investigate its significance in diagnosis of MDS.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Expression of CD71 on cell proliferation in hematologic malignancy and its correlation with Ki-67 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to explore the expression of CD71, as a proliferation indicator, on cell proliferaration in hematologic malignancy and its correlation with Ki-67, so as to assess the feasibility of CD71 instead of Ki-67 for assaying cell proliferation by flow cytometry (FCM).</p><p><b>METHODS</b>(1) Compared with mature B lymphoctyes during stationary phase in peripheral blood from healthy people, the cell cycle and the expression of CD71 and Ki-67 of cell lines from patients with leukemia and lymphoma were examined, the correlation among CD71, S-phase cell fraction (SPF) and Ki-67 were analyzed; (2) Compared with mature B lymphoctyes in bone marrow from non-hematologic disease patients, the expression and correlation of CD71 and Ki-67 of all kinds of leukemic cells and myeloma cells from bone marrow were analyzed by using Ki-67/CD71/CD45/CD123, Ki-67/CD71/CD45/CD20 or Ki-67/CD71/CD45/CD138.</p><p><b>RESULTS</b>(1) in respect to the expression rate of CD71 on tumor cell lines, the expression rate of CD71 on HL-60 cells was (99.77 ± 0.064)%, the expression rate of CD71 on NB4 cells was (99.23 ± 0.12)%, the expression rate on THP-1 cells was (98.90 ± 0.30)% and the expression rate on K562 cells was (97.03 ± 0.15)% in myelogenous leukemia cell lines, the expression rate of CD71 on Raji cells was (99.35 ± 0.21)% and the expression rate on Mino cell was (96.95 ± 0.42)% in lymphoma cell lines, which were also obviously higher than that on cells of the control group (P < 0.05); (2) in respect to the expression rate of CD71 on tumor cells in bone marrow, the expression rate of CD71 on poorly differentiated AML(M1 and M2) cells was (51.50 ± 19.31)%, the expression rate of CD71 on acute promyelocytic leukemia (AML-M3) cells was (35.71 ± 14.02) %, the expression rate of CD71 on acute monocytic leukemia (AML-M5) cells was (30.54 ± 14.38)%, the expression rate of CD71 on acute T lymphoblastic leukemia cells was (68.40 ± 20.83)%, the expression rate of CD71 on acute B lymphoblastic leukemia was (39.67 ± 18.27)%, the expression rate of CD71 on multiple myeloma (MM) cells was (55.49 ± 18.15%), the expression rate of CD71 on chronic lymphocytic leukemia(CLL) was (1.32 ± 0.33%), which were also higher than that on cells in the control group(P < 0.05) except for CLL cells (P > 0.05); (3) CD71 had a positive linear corrlation with SPF in cell lines (r = 0.914, P < 0.05), and also had a positive linear corrlation with Ki-67 in cell lines and carcinoma cells from bone marrow (r = 0.894,r = 0.904, P < 0.05).</p><p><b>CONCLUSION</b>The CD71 can take the place of Ki-67 as an indicator of cell proliferation activity of hematologic malignancies and the determination CD71 by FCM is simpler and better than that of Ki-67 in respest of methodology.</p>

Expression of CD58 in childhood B-lineage acute lymphoblastic leukemia and its feasibility in minimal residual disease detection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

Down-regulatory effect of Nucleostemin expression on signal molecule of PI3K/AKT/mTOR pathway in HL-60 cells / 中国实验血液学杂志

This study was purpose to explore the down-regulatory effect of nucleostemin (NS) expression on signal molecules of PI3K/AKT/mTOR pathway belonged to candidate ways of p53-independent signal pathway in the leukemia cells. The expression of NS was interfered by using recombinant lentivirus expression vector NS-RNAi-GV248 to transfect HL-60 cells of p53 deficiency. The expression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by using Real-time PCR. The results of showed that the HL-60 cells were transfected by recombinant lentivirus vector NS-RNAi-GV248 successfully and with transfection rate up to 80%. According to results of Real-time PCR detection, the inhibition rate of NS gene was 56.5% in HL-60 cells. And the expression levels of PI3K,AKT and GβL mRNA (0.491 ± 0.084,0.398 ± 0.164, 0.472 ± 0.097 respectively) were obviously down-regulated by silencing NS, and showed statistical difference (P < 0.05) in comparison with control (1.002 ± 0.171, 1.000 ± 0.411, 1.001 ± 0.206 respectively) . It is concluded that the changes of signal molecules of PI3K/AKT/mTOR pathway positively correlate with NS down-regulation, which provides evidence for confirming PI3K/AKT/mTOR signal pathway possible as a type of NS p53-independent pathway.

Effects of silent nucleostemin gene expression on apoptosis of HL-60 cells in vitro / 中国实验血液学杂志

This study was aimed to explore whether the apoptosis of leukemia cells can be induced by targeting silencing nucleostemin gene in vitro. HL-60 cells were taken as the model, and were directly transfected with nucleostemin short hairpin RNA (NS-shRNA). Sequences unrelated with NS gene were taken as control. The blocking effect of NS-shRNA was detected by RT-PCR, the morphology changes in living cells were observed under inverted microscope, and the changes of cell shape and nucleus were detected by Wright-Giemsa staining. The amount of apoptotic cells were assayed by flow cytometer (FCM) and TUNEL technique, and the positive rate of apoptosis was determined meanwhile. The results showed that two NS-shRNA were synthesized in vitro, and the more effective one was selected to be transfected into HL-60 cells. The blocking rate of NS-mRNA reached to 74.94%. After transfection for 48 hours, Wright-Giemsa staining showed nuclear fragmentations and "apoptosis body" in cells. The apoptosis rate in transfected group detected by flow cytometry and TUNEL method were (25.32 +/- 3.06)% and (27.3 +/- 3.21)% respectively, but were only (3.12 +/- 0.38)% and (3.30 +/- 1.52)% in control group, the difference between the transfected group and the control group was significant (p < 0.01). It is concluded that the apoptosis of HL-60 leukemia cells can be induced by the silencing NS gene expression in vitro, which provides a theoretical basis for using NS gene as a candidate target gene in therapy of malignant tumor.