Activated interferon response from DNA damage in multiple myeloma cells contributes to the chemotherapeutic effects of anthracyclines.

Introduction: Multiple myeloma (MM) is a malignant plasma cell disease caused by abnormal proliferation of clonal plasma cells in bone marrow. Upfront identification of tumor subgroups with specific biological markers has the potential to improve biologically-driven therapy. Previously, we established a molecular classification by stratifying multiple myeloma into two subtypes with a different prognosis based on a gene module co-expressed with MCL-1 (MCL1-M). Methods: Gene Ontology (GO) analysis with differentially expressed genes was performed to identify signal pathway. Drug sensitivity was analyzed using the OncoPredict algorithm. Drug sensitivity of different myeloma cell lines was detected by CCK8 and flow cytometry. RNA-seq was performed on drug-sensitive cell lines before and after adriamycin treatment. RT-qPCR was used to further verify the sequencing results. The expression of γ-H2AX and dsDNA in sensitive and resistant cell lines was detected by immunofluorescence method. Results: In our study, we demonstrated that MCL1-M low MM were more sensitive to anthracyclines. We treated different myeloma cell lines with doxorubicin in vitro and discovered the association of drug sensitivity with IFN signaling. Herein, we demonstrate that the doxorubicin-sensitive myeloma cell line showed significant DNA damage and up-regulated expression of genes related to the IFN response, which was not observed in drug-insensitive cell lines. Discussion: Our results suggest that the active IFN signaling pathway may serve as a marker for predicting chemotherapy sensitivity in patients with myeloma. With our MCL1-M molecular classification system, we can screen patients with a potentially good response to the interferon signaling pathway and provide individualized treatment for MM. We propose IFN-a as adjuvant therapy for patients with myeloma sensitive to anthracyclines to further improve the therapeutic effect and prolong the survival of patients.

[Expression of CD30 in Patients with Diffuse Large B-Cell Lymphoma and Clinical Significance].

OBJECTIVE: To investigate the expression and clinical significance of CD30 in patients with diffuse large B-cell lymphoma (DLBCL). METHODS: A retrospective analysis was conducted on 124 cases of primary DLBCL diagnosed at Changzhou Second People's Hospital Affiliated with Nanjing Medical University from January 2018 to July 2020. The expression of CD30 in patients with DLBCL was detected by immunohistochemical method, and the clinicopathological characteristics were analyzed and compared between CD30+ and CD30- groups. Kaplan-Meier analysis was used for survival analysis. The relationship between CD30 expression and clinical features and prognosis were analyzed. RESULTS: Among the 124 patients with DLBCL, 19 patients expressed CD30, and the positive rate is 15.32%. The clinico-pathological characteristics of CD30+ in patients with DLBCL were characterized by low age, more common in males, fewer extranodal lesions, lower international prognostic index (IPI), GCB type being more common in Hans subtype, and achieving better therapeutic effects (P < 0.05). However, there were no significant statistical differences in B-symptoms (P =0.323), Ann Arbor staging (P =0.197), Eastern Cooperative Oncology Group (ECOG) score (P =0.479), lactate dehydrogenase (LDH) (P =0.477), and the involvement of bone marrow (P =0.222). There were significant differences in OS and PFS between the CD30+ and CD30- groups (χ2=5.653, P =0.017; χ2＝4.109，P =0.043), the CD30+ group had a better prognosis than that of the CD30- group. The results of subgroup analysis showed that the CD30+ group in the IPI score=1-2, LDH elevated group had a better prognosis (P < 0.05). In the subgroups of Ann Arbor staging III-IV (P =0.055) and non GCB type (P =0.053), the CD30+ group had a good prognosis trend, but the difference was not statistically significant. The results of univariate analysis showed that the good prognosis of DLBCL patients was closely related to CD30+ expression, no B-symptoms, early Ann Arbor staging, low ECOG score, normal LDH, low IPI score, fewer extranodal involvement, and obtaining the best therapeutic effect as CR (all P <0.05). COX multivariate regression analysis showed that the presence of B-symptoms and achieving the best therapeutic effect as Non-CR were independent risk factors affecting the prognosis of DLBCL patients (P < 0.05). CONCLUSION: The CD30+ expression in DLBCL patients indicates a good prognosis and has certain diagnostic value in evaluating the prognosis of DLBCL patients.

Molecular genetic and clinical characterization of acute myeloid leukemia with trisomy 8 as the sole chromosome abnormality.

INTRODUCTION: The aim of the study was to determine molecular genetic and clinical characterization of acute myeloid leukemia (AML) with trisomy 8 as the sole chromosome abnormality, a recurrent but rare chromosomal abnormality in AML. METHODS: Interphase fluorescence in situ hybridization, reverse transcriptase-quantitative polymerase chain reaction for gene rearrangement and next-generation sequencing (NGS) were performed on sole trisomy 8 AML patients. RESULTS: A total of 35 AML patients with trisomy 8 as the sole chromosome abnormality were screened. The most frequently mutated genes were DNMT3A(37.1%), RUNX1(28.6%), FLT3-ITD(28.6%), IDH2(22.9%), NPM1(17.1%), and ASXL1 (14.3%). The sole +8 AML patients exhibited more mutations in RUNX1 (28.6% vs. 4.8%, P = 0.001) and ASXL1 (14.3% vs. 4.8%, P = 0.039) by comparing with normal karyotype AML (NK AML) patients(n = 63). The sole +8 AML patients(n = 35) with RUNX1 or IDH2 mutations showed significantly lower WBC counts, while FLT3-ITD showed higher white blood cell (WBC) counts as compared to the corresponding wild-type groups. Total of 45.7% patients achieved complete remission (CR) after the first induction therapy. The CR rate of patients with FLT3-ITD or IDH1 mutation was significantly lower than that in the corresponding wild-type cases (P = 0.047, 0.005, respectively). The median overall survival (OS) and disease-free survival (PFS) were 18.0 (95% CI: 10.8-25.2) and 10 (95% CI: 6.7-13.3) months, respectively. FLT3-ITD mutations and allogeneic hematopoietic stem cell transplantation (allo-HSCT) were independent prognostic markers for OS in multivariable analysis. CONCLUSION: The results suggest a possible association between trisomy 8 and additional mutations that may influence clinical feature and prognosis.

PTPN11 mutations in adult acute myeloid leukaemia: Prevalence and clinical implications in the context of NPM1 mutation.

More than 90% of NPM1-mutated acute myeloid leukaemia (NPM1mut AML) patients have been determined to harbour other known concurrent mutations. However, there is limited data on the frequency of PTPN11 and its clinical impact in NPM1mut AML. Next-generation sequencing(NGS) was performed retrospectively on 112 genes in 254 patients with NPM1mut AML. Overall, 33 PTPN11 mutations were detected in 29 of the 254 patients (11.42%) screened. PTPN11 alterations were exclusively missense mutations affecting residues located within the N-SH2 (n = 25) and PTP (n = 8) domains and clustered overwhelmingly in exon 3 (n = 25). NPM1mut AML patients with mutant PTPN11 had significant associations with mutations in epigenetic regulators(TET2, IDH1/2, and DNMT3A) (72.41% vs. 46.67%, P = 0.009) and cohesins (RAD21,SMC1A, and SMC3)(10.34% vs. 1.33%, P = 0.02) compared to patients with wild-type PTPN11. Among the patients treated with intensive chemotherapy, the differences in disease-free survival (DFS) and overall survival (OS) between those with mutant and wild-type PTPN11 were not significant (P = 0.4, 0.83, respectively). In conclusion, PTPN11 mutations were frequently identified in NPM1mut AML patients and clustered in exon 3. PTPN11 mutations more frequently co-occurred with mutations involving epigenetic regulators but had no impact on prognosis.

Companion gene mutations and their clinical significance in AML with double or single mutant CEBPA.

INTRODUCTION: We report the co-mutations in AML with CEBPAsm or CEBPAdm and their clinical features in a large cohort (n = 302) of CEBPAmut AML patients. MATERIALS AND METHODS: We retrospectively sequenced 112 genes in 302 patients with CEBPAmut using NGS, and studied the spectrum and clinical impact of co-mutations in CEBPAdm and CEBPAsm. RESULTS: ① The average number of mutations in CEBPAsm and CEBPAdm AML was comparable, but not significant (P = 0.17). ② CEBPAdm patients exhibited more mutations in CSF3R (P = 0.037), GATA2 (P = 0.022), and WT1 (P = 0.046). In contrast, CEBPAsm patients more frequently harbored mutations in NPM1 (P = 0.000), FLT3-ITD (P = 0.025) and NOTCH2 (P = 0.043), as well as mutations in signaling pathways and spliceosomes (P = 0.064, P = 0.027, respectively). ③ Patients with CEBPAsm/TET2mut or CEBPAsm /GATA2mut had higher platelet counts (both P = 0.011), while patients with CEBPAdm /TET2mut had significantly higher hemoglobin levels (P = 0.009). The CR rate of patients with FLT3-ITD mutations was significantly lower in the CEBPAsm group than the CEBPAdm group (P = 0.028). CONCLUSIONS: CEBPAsm and CEBPAdm AML are each associated with their own complex co-mutation cluster. Some co-mutations influence the clinical features and CR rate differently in patients with different CEBPA mutational status.

Adult Acute Myeloid Leukemia with the KMT2A-Mixed Lineage Leukemia T10 Fusion: An Analysis of 10 Cases Showed Common Features and Frequent Mutations in the RAS Signaling Pathway.

Mixed lineage leukemia (MLL) T10 is a relatively rare partner for the KMT2A lysine (K)-specific methyltransferase 2A gene. The common features and coexisting mutations of acute myeloid leukemia (AML) patients with KMT2A-MLLT10 remain unknown. In this study, 10 adult AML patients with KMT2A-MLLT10 fusions were picked up from 496 AML patients by using RT-polymerase chain reaction (PCR) and/or fluorescence in situ hybridization, and then screened for mutations in the 49 genes panel with next-generation sequencing and PCR, followed by direct Sanger sequencing. Of the 10 unique individuals identified, 6 were male and 4 were female (M:F ratio, 1.5:1) with ages ranging from 19 to 52 years (median 39.5 years). Most (90%, 9/10) patients with KMT2A-MLLT10 were accompanied by additional mutations. Twelve mutated genes were detected, averaging 2.1 mutations per patient (range, 0-4). The most frequently mutated gene was NRAS (n = 5). Clinical and laboratory data pointed to common features: French American British-M5 subtype (n = 7), a high rate of relapse, and biomarkers CD33 (n = 10), CD117 (n = 9), CD13 (n = 8), and CD64 (n = 8). Overall, most patients harbored at least one mutation. A high incidence of mutations affecting the RAS signaling pathway or RAS regulating components was found in 50% (5/10) patients. The overall survival is about 12.0 months. Allogeneic-hematopoietic stem cell transplantation trends to improve survival in selected patients.

Mutational landscape of chronic myelomonocytic leukemia and its potential clinical significance.

We evaluated the mutational landscape of chronic myelomonocytic leukemia (CMML) and its potential clinical significance. We analyzed 47 samples with a panel of 112 genes using next-generation sequencing. Forty-five of the 47 patients (95.74%) had at least one mutation identified, with an average of 3.7 (range 0-9) per patient. The most common mutation was NRAS, followed by ASXL1, TET2, SRSF2, RUNX1, KRAS, and SETBP1. Patients 60 years and older more frequently had mutations in TET2 (56% vs. 9.09%, P = 0.001) and ASXL1 (48% vs. 18.18%, P = 0.031) than patients younger than 60 years. Median overall survival (OS) in patients with CMML was 22.0 months (95% CI 19.7-24.3 months). ASXL1 (18 vs. 22 months, P = 0.012), RUNX1 (17 vs. 22 months, P = 0.001), and SETBP1 (20 vs. 27 months, P = 0.032) mutations predicted inferior OS. However, only RUNX1 mutation was significantly associated with inferior acute myeloid leukemia (AML)-free survival. Our data showed that mutation profile differed significantly between CMML patients aged 60 years and older versus those younger than 60 years, and some of these mutations impact the progression and prognosis of the disease to a certain extent.

[Mechanism of Anti Apoptosis and Immune Evasion in Drug-Resistant Leukemia Cells Mediated by STAT3].

OBJECTIVE: To investigate the mechanisms of anti-apoptosis and immune evasion in drug-resistant leukemia cells mediated by STAT3, further to explore the possible mechanism of leukemia relapse caused by minimal residual. METHODS: Drug-resistance leukemia cell line was established by transfecting pcDNA3.1-STAT3 into K562 cells (K562/STAT3). The expression of STAT3, BAX and NKG2D ligands (MICA and ULBP1) in K562/-cells, K562/STAT3 were detected by Western blot and/or RQ-PCR. Cells apoptosis and the killing effect of NK cells on leukemia cells were detected by flow cytometry. RESULTS: The expression of the total STAT3, STAT3 phosphorylation in K562/STAT3 was significantly increased, and P-gp mRNA expression was increased also significantly (P<0.005). In K562/STAT3 cells, the expression of pro-apoptotic BAX (P=0.005) was significantly lower, and the number of apoptotic cells (P=0.002) induced by adriamycin was significantly decreased as compared with those in K562/- cells. After K562/STAT3 cells were treated by STAT3 inhibitor (SH-4-54), the expression of BAX mRNA (P=0.017) was significantly higher and the number of apoptotic cells (P=0.005) was significantly increased. The MICA and ULBP1 mRNA expression in K562/STAT3 cells was significantly lower than that in K562/- cells, and also for MICA and ULBP1 protein (MICA and ULPB1 mRNA: P<0.0001, MICA protein: P=0.001, ULPB1 protein: P=0.022). After K562/STAT3 cells were treated with STAT3 inhibitor (SH-4-54), the expression of MICA mRNA and protein was increased (mRNA: P=0.001, protein: P=0.002), but ULBP1 mRNA and protein showed no significantly change (mRNA: P=0.137, protein: P=0.1905). The cytotoxicity of NK cells to K562/STAT3 cells was susceptible as compared with K562/- (P=0.002), but the cytotoxicity of K562/STAT3 cells to NK cell could be recovered by STAT3 inhibitor (P=0.006). CONCLUSION: STAT3 phosphorylation can inhibits cell apoptosis and promotes cell immune escape. STAT3 inhibitors can promote the apoptosis of leukemia cells and increase their sensitivity to NK cells.

[Analysis of RUNX1 Gene Mutation in Patients with Myelodysplastic Syndrome].

OBJECTIVE: To investigate the mutation of RUNX1 gene in patients with myelodysplastic syndrome (MDS) and its correlation with other gene mutations and some clinical parameters. METHODS: The mutations of RUNX1, DNMT3A, TET2, IDH1/2, NPM1, FLT3-ITD and C-KIT in 170 patients with MDS were detected by direct and indirect sequencing of genomic DNA-PCR amplification products. RESULTS: The RUNX1 mutation was found in 23 patients (13.5 %, 23/170). Among the 170 patients, other most frequent mutation was TET2 (11.2%, 19/170), followed by mutations in DNMT3A (9.4%, 16/170), NPM1 (8.2%, 14/170), IDH2 (4.1%, 7/170)、FLT3-ITD (2.9%, 5/170), IDH1 (1.7%, 3/170) and c-KIT (0.58%, 1/170). The most common coexisting mutations were TET2 (5/23). The RUNX1-mutated group showed significantly higher leukocyte levels, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet counts in comparison with RUNX1 non-mutation group (P＜0.05). whereas there were no statistically significant difference in age, MDS subtype, karyotype and hemoglobin level between 2 groups (P＞0.05). Seventeen patients harboring RUNX1 mutations were followed up and almost 47.05% (8/17) of the patients progressed into acute myeloid leukemia (AML). The rates of transformation into AML in ASXL1-mutation group was significantly higher than that in ASXLL- non-mutation group (47.05% vs 11.7%) (P=0.001). CONCLUSION: The incidence of RUNX1 mutation is high in MDS patients. The RUNX1-mutated patients have higher leukocyte level, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet count.

Preparation and anti-Raji lymphoma efficacy of a novel pH sensitive and magnetic targeting nanoparticles drug delivery system.

BACKGROUND: Non-Hodgkin's lymphoma (NHL) is a heterogeneous class of cancers that arises in lymph nodes or other lymphatic tissues, which causes many deaths worldwide and its incidence is increasing. METHODS: In this study, a pH-responsive DMSA-Fe3O4 magnetic nanoparticles (MNPs) covalently connect with ADM and As2O3 as a drug delivery system was invented to discuss the anticancer efficacy in non-Hodgkin's lymphoma (NHL) cell line--Raji. RESULTS: Detailedly, according to the chelation of ADM and Fe2+, the release rate of ADM was accelerated in acidic environment, and slowed down/blocked in neutral environment. The inhibitory effect to induce apoptosis of Fe3O4/As2O3+Doxil on Raji cells was obvious compared with that of single-drug group. Furthermore, the expression of Bcl-2 gene in Raji cells was suppressed under the action of MNPs. CONCLUSION: Taken together, the novel pH-responsive MNPs was proven to be a promising synergistic form of magnetic targeted drugs for clinical treatment of human Raji lymphoma.

[Coexisting Mutations in IDH1/2-Mutated Acute Myeloid Leukemia].

OBJECTIVE: To explore the coexisting mutations in IDH-mutated acute myeloid leukemia(AML) and its relation with partial clinical parametrs. METHODS: The exon 4 mutation of IDH1/2 gene was screened by using genome DNA-PCR combined with sanger sequencing, 51 targeted gene mutations in the patients with IDH1/2 mutation were detected by using high throughput DNA sequencing combined with sanger sequencing. RESULTS: Among 358 patients, the IDH1/2 mutation was found in 46 cases including IDH1 mutation in 35 cases and IDH2 mutation in 11 cases, 97.87%(45/46) patients with IDH1/2 mutation simultaneously carried other gene mutations including 8(17.8%) cases with mutation of double gene, 17(37.8%) cases with mutation of 3 genes and 20(44.4%) cases with mutation of ≥ 4 genes. The mutation frequency of each patient averaged 3.52 times. In mutation of accompanied genes, the common genes were NPM1(n=29, 63.0%), next DNMT3A(n=25, 54.3%), FLT3-ITD(n=7, 15.2%), TET2(n=5, 10.9%) and NRAS(n=5, 10.9%). The average WBC level of patients with NPM1 mutation in IDH1 mutation group was higher than that of patients in wild type group(P<0.05). The complete remission (CR) rate of patients with DNMT3A mutation was significant lower than that of patients with wild type (30% vs 80%, P<0.01). The presence of ≥ 4 mutations was found to be significantly associated with higher white blood level than that in the patients with double mutations(P<0.05). CONCLUSION: More than 95% AML patients with IDH1/2 mutation commonly show additional mutations. The number and the type of IDH coexisting mutations have certain effect on the clinical features and CR rate.

STAT3 signaling pathway is involved in decitabine induced biological phenotype regulation of acute myeloid leukemia cells.

OBJECTIVE: This study aimed to investigate the role of signal transduction and transcriptional activator STAT3 and relevant signaling pathway in the DAC regulated biological phenotype of AML cells. METHODS: The effect of DAC at different concentrations on the proliferation of HL-60 cells was determined. After DAC treatment for 48 h, the killing capability of NK cells against HL-60 cells and the protein expressions of STAT3, JAK1, JAK2, SOCS-1 and SOCS-3 were evaluated. RESULTS: DAC markedly inhibited the proliferation of HL-60 cells. After the treatment of 48 hr with 0.2, 0.5 and 1.0 mol/L DAC, the HL-60 viability was reduced by 25±13%, 39±8% and 50±7% (P<0.01), respectively, and the early apoptosis rate was increased to 24.77±7.5%, 27.1±4.48% and 30.53±3.93%, respectively (control: 3.11±0.12%, P<0.01). DAC up-regulated the expression of MICA/B, ULBP-1 and ULBP-3 in HL-60 cells, and increased the killing activity of NK cells to HL-60 cells. DAC significantly induced the apoptosis of HL-60 cells and up-regulated the expression of NKG2D ligands in a dose dependent manner. Western blot assay showed the protein expression of STAT3, JAK, JAK2, phosphorylated STAT3, phosphorylated JAK1 and phosphorylated JAK2 decreased, while that of SOCS-1 and SOCS-3 increased in HL-60 cells after DAC treatment. CONCLUSION: In HL-60 cells, DAC can markedly inhibit their proliferation and up-regulate the expression of NKG2D ligands, and DAC also increase the cytotoxicity of NK cells to HL-60 cells, which may be related to the STAT3 related signaling pathway.

Matrine increases NKG2D ligand ULBP2 in K562 cells via inhibiting JAK/STAT3 pathway: a potential mechanism underlying the immunotherapy of matrine in leukemia.

PURPOSE: The study aimed to investigate the role of the JAK/STAT3 pathway in the matrine induced ULBP2 expression on the human chronic myelogenous leukemia K562 cells. METHODS: K562 cells were cultured, and the relevant mRNA expressions were detected. RESULTS: Matrine induced the expression of four NKG2D ligands on K562 cells, of which ULBP2 had the highest increase. After treatment with 0.8 mg/mL matrine for 24 h, the mean fluorescence intensity (MFI) of ULBP2 increased. After matrine treatment, the sensitivity of K562 cells to NK cell-mediated killing increased significantly. After treatment with 0.2, 0.5 and 0.8 mg/ mL matrine, the percentage of K562 cells killed by NK cells was significantly higher than that of untreated cells (29.2%) (P<0.05). Matrine significantly inhibit the protein expression of phosphorylated STAT 3 and JAK2. Matrine markedly inhibited the IL-6 expression of K562 cells, and antagonized the IL-6 mediated STAT3 and JAK2 phosphorylation. In addition, matrine enhanced the inhibitory effect of STAT 3 inhibitor on STAT 3 activity. The silencing of STAT expression and inhibition of STAT3 activity significantly up-regulated the ULPB2 expression. Matrine had no effect on the expression of IL-6R and gp130 on K562 cells, the mRNA expression of IL-6R and gp130 increased slightly and the sgp 130 in cell supernatant significantly increased. CONCLUSIONS: Our findings reveal IL-6 and IL-6 receptor-mediated JAK/STAT3 pathway is involved in the matrine induced up-regulation of NKG2D ligands ULBP2 on K562 cells. Matrine might inhibit IL-6 expression and then suppress the activation of IL-6 receptor-mediated JAK/STAT3 pathway.

[Study of NK cells dysfunction in multiple myeloma patients].

OBJECTIVE: To explore the mechanism of NK cell dysfunction in patients with multiple myeloma (MM). METHODS: The expression of inhibitory receptors (CD158a and CD158b) and activating receptors NKG2D and NCRs (NKp30, NKp44 and NKp46) on CD3-CD56+NK cell of 13 MM patients and 30 healthy controls were analyzed by flow cytometry. The concentration of soluble NKG2D ligands (MICA, MICB, ULBP1, ULBP2 and ULBP3) in serum was detected by enzyme- linked immunosorbent assay (ELISA), and the cytotoxicity of NK cell against MM cell line by flow cytometry. RESULTS: There are no significant differences of percentage and absolute number of NK cells, and the expression level of CD158a and CD158b between MM patients and healthy individuals (P>0.05). No NKp44 expression was detected on fresh isolated NK cells from both groups. There is no difference in inhibitor receptors expression between MM patients and healthy individuals but the expression of NKG2D, NKp30 and NKp46 on NK cells were higher in MM patients as compared with that in healthy individuals. The concentration of soluble NKG2D ligands in serum was higher in MM patients as compared with that in healthy individuals (P<0.05). Cultured healthy individual's NK cells with MM patient's serum could significantly decrease its cytotoxicity against MM cell line U266 cells [(38.5 ± 6.5) % vs (25.4 ± 5.9)%, P=0.044ï¼½. CONCLUSION: The higher level of soluble NKG2D ligands in serum may be the mechanism of NK cell dysfunction in MM patient.

STAT3 contributes to NK cell recognition by modulating expression of NKG2D ligands in adriamycin-resistant K562/AO2 cells.

Leukemic cells can survive after chemotherapy by acquisition of multidrug resistance genes, but other phenotypes related to escape from immune recognition remain elusive. Adriamycin-resistant K562/AO2 cells are less susceptible to elimination by NK cells compared with wild type K562 cells due to lower expression of NKG2D ligands. Treatment of K562/AO2 cells with STAT3 inhibitor VII resulted in reduced expression of multidrug resistance gene P-glycoprotein, and up-regulation of NKG2D ligands on K562/AO2 cells. Meanwhile, K562/AO2 cells treated with STAT3 inhibitor proliferated less and were more susceptible to killing by NK cells than untreated K562/AO2 cells. The enhanced cytotoxicity of NK cells against K562/AO2 cells was partly blocked by treatment of NK cells with anti-NKG2D antibodies. These data suggest that STAT3 contributes to NK cell recognition by modulating NKG2D ligands in K562/AO2 cells, which may a mechanism by which cells survive and cause relapse of leukemia.

[Enhanced cytotoxicity against leukemia cells of natural Killer cells from cord blood after expansion in vitro].

OBJECTIVE: To investigate the enhanced cytotoxicity against leukemia cells of natural Killer (NK) cells from cord blood (CB) after expansion in vitro. METHODS: NK cells was expanded on a layer of trophoblast cells with irradiated K562-mb15-41BBL cell line for 21 days. The levels of receptors on NK cells were detected by flow cytometry. Cytotoxicity of expanded NK cells against leukemia cells and specific ligand of immunoglobulin like(Ig- liKe)receptors were assessed using 51Cr released assay. RESULTS: There were no differences of inhibitory receptors expression between fresh NK cells and expanded NK cells [CD158a:(16.77±11.65)% vs(14.37±11.12)%, P>0.05; CD158b: (42.48±18.11)% vs (40.92±19.02)%, P>0.05; NKG2A: (70.20±18.43)% vs (78.90±13.69)%, P>0.05], but higher activated receptors expression on expanded NK cells [NKp30: (54.10±13.27)% vs (4.14±2.05)%, P<0.05; NKp44: (72.10±17.30)% vs (0.52±1.16)%, P<0.05; NKp46: (80.63±14.01)% vs (44.19±6.19)%, P<0.05; NKG2D: (97.50±2.55)% vs (72.25±14.35)%, P<0.05]. Expanded NK cells showed higher cytotoxicity against leuKemia cell lines than fresh NK cells [K562: (74.3±3.6)% vs (55.3±4.2)%, P<0.05; Raji: (60.6±5.0)% vs (12.0±3.6)%, P<0.05]. CD158a⁻ CD158b⁻ NK cells had higher cytotoxicity on four types of target cells, but CD158a⁺CD158b⁻ CB-NK cell had lower cytotoxicity on 221-Cw4 and 221-Cw3Cw4 cells. CD158a⁻ CD158b⁺ CB- NK cells had lower cytotoxicity on 221-Cw3 and 221-Cw3Cw4, but CD158a⁺CD158b⁺ CB-NK cells had higher cytotoxicity on 721- 221 cells. CONCLUSION: Expression of activated receptors of expanded NK cells were up-regulated, but no changes of inhibitory receptors. Expanded NK cells showed high cytotoxicity against leukemia cells and kept the specificity of ligand of Ig-like receptors, which could be beneficial to cell-therapy for tumor.

[Roles of NKG2D in cytokine-induced killer (CIK) against hematological malignant cells lines].

This study was purposed to investigate the CIK cell cytotoxicity to hematological malignant cell lines by interaction NKG2D receptors and corresponding ligands. The CIK cells was expanded from healthy individual with interferon (IFN)γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2). The subset of lymphocyte and the expression of NK cell receptors on CIK cells was detected by flow cytometry; NKG2D ligand expression on hematological malignant cell lines was also analyzed by flow cytometry, the calcein acetoxymethyl ester (CAM) was used for labeling target cells, then the cytotoxicity of CIK cells to hematological malignant cell lines was detected by flow cytometry. The results showed that most of CIK cells expressed CD3 (97.85 ± 1.95%) , CD3(+)CD8(+) cells and CD3(+)CD56(+) cells increased significantly as compared with un-cultured cells (P < 0.001;P = 0.033). About 86% CIK cells expressed NKG2D receptor but no other NK receptors such as CD158a, CD158b and NCR. Different levels of NKG2D ligands were detected in hematological malignant cell lines U266, K562 and Daudi. CIK cells showed high cytotoxicity to these three different cell lines, and this cytotoxicity was partially blocked by treating CIK cells with anti-NKG2D antibody (U266 52.67 ± 4.63% vs 32.67 ± 4.81%, P = 0.008;K562 71.67 ± 4.91% vs 50.33 ± 4.91%, P = 0.007;Daudi 68.67 ± 5.04 vs 52.67 ± 2.60%, P = 0.024) . It is concluded that most of CIK cells express NKG2D receptor, interaction of NKG2D-NKG2D ligands may be one of the mechanisms, by which CIK cells kill hematological malignant cells.

[Analysis of IDH1 and IDH2 mutations in patients with acute myeloid leukemia].

OBJECTIVE: To explore the prevalence of IDH gene (IDH1 and IDH2) mutations, types of mutations in patients with acute myeloid leukemia (AML), correlation with the internal tandem duplication(ITD) mutation of FLT3 gene, NPM1 gene mutation and some clinical characteristics. METHODS: The mutations of IDH1 and IDH2 gene at exon 4, NPM1 gene at exon 12 and FLT3-ITD at exon 14 and 15 in 163 newly diagnosed AML patients were detected by PCR amplification followed by direct sequencing of genomic DNA. RESULTS: (1) IDH mutations were found in 25 patients (25/163), and all were heterozygous, of which IDH1 in 7 patients (4.29%) and IDH2 in 18 (11.04%). A total of 4 types of IDH1 mutations were identified (c.395G→A, p.R132H, n = 4; c.394C→A, p.R132S, n = 1; c.394C→G, p.R132G, n = 1; c.315C→T, n = 1). The IDH1 mutation caused substitutions of residue R132 except for one (c.315C→T). All IDH2 mutations caused changes of R140 (c.419G→A, p.R140Q, n = 18). The incidence of IDH2 mutation was significantly higher than that of IDH1 mutation (11.0% v 4.3%, P = 0.022). Both IDH1 and IDH2 mutation were detected in one patient, while IDH1 was synonymous substitution (c.315C→T). IDH-mutated cases showed a significantly higher frequency of concurrent FLT3-ITD mutation compared with wildtype cases (34.6% vs 11.9%, P = 0.003), so did IDH mutations concurrent NPM1 mutation vs NPM1 wildtype (28.1% vs 12.7%, P = 0.033), of which the frequency of concurrent NPM1 and FLT-ITD mutations cases with the IDH mutation was significantly higher than that of NPM1 and FLT-ITD negative (45.5% vs 11.7%, P = 0.002). IDH mutation incidence was significantly higher in normal karyotype cases than in abnormal ones (20.5% vs 5.8%, P = 0.020). Patients with IDH mutations were significantly older than wildtype patients(P < 0.001), whereas, there were no statistically significant differences in gender, peripheral blood (PB) count at diagnosis between two groups. CONCLUSIONS: The incidence of IDH mutation is higher in patients with de novo AMLs, of which IDH2 mutation more frequently, and the patients associated with older age, normal karyotype at diagnosis. IDH mutation has a strong association with NPM1 and FLT3-ITD mutations, suggesting that IDH mutation has synergistic effect with the latter gene on leukemogenesis.

P53 deletion is independently associated with increased age and decreased survival in a cohort of Chinese patients with diffuse large B-cell lymphoma.

The purpose of the study was to compare cytogenetic profiles and survivals between elderly and non-elderly Chinese patients with diffuse large B-cell lymphoma (DLBCL). We identified 50 patients with DLBCL and divided them by age into elderly (≥60 years) and non-elderly (< 60 years) groups. We detected deletion of P53 or translocations in Bcl-2, Bcl-6 or c-myc genes by fluorescence in situ hybridization (FISH). P53 deletion was significantly more common in elderly versus non-elderly patients (62% vs. 17%, respectively, p=0.001) There were no significant differences in rates of Bcl-2, Bcl-6 or c-myc gene rearrangements between elderly and non-elderly patients (p>0.25 for each). Median survival was significantly longer in non-elderly compared to elderly patients. P53 deletion was an independent prognostic factor for decreased survival in patients with DLBCL, independent of age. In conclusion, P53 deletion as detected by FISH is associated with both increased age and decreased survival in Chinese patients with DLBCL. This association with decreased survival is at least partly independent of age. P53 deletion could serve as a prognostic factor in DLBCL independent of or in combination with age.