Objective: To investigate the effects of different doses of X ⁃rays irradiation combined with allogeneic lymphocyte infusion on the establishment of aplastic anemia in mice. Methods: Forty BALB/c mice were randomly divided into four groups : the 3 Gy group (n = 9) , the 4 Gy group (n = 9) , the 5 Gy group (n = 10) , and the con⁃trol group (n = 12) . In the 3 Gy , 4 Gy , and 5 Gy groups , the experimental mice were exposed to corresponding do⁃ses of X ⁃ray and then intravenously infused with 0. 2 mL mixed suspension of the thymus and spleen cells from DBA/2 mice , at a concentration of 1 × 107 cells/mL , within 4 hours after irradiation. The control group did not un⁃dergo X ⁃ray irradiation and infused with an equivalent volume of physiological saline instead. Blood samples were collected from the orbital venous plexus of BALB/c mice and analyzed using an animal automated hematology analy⁃zer to measure peripheral blood parameters , including red blood cells ( RBC) , white blood cells ( WBC) , and platelets (PLT) . The general condition of mice was monitored daily , and survival rates were recorded for each group. At the experimental endpoint , the tibias were harvested for hematoxylin and eosin (HE) staining , while the femurs were used to prepare bone marrow smears for morphological examination. For the 5 Gy group , T ⁃cell subsets(ELISA) at the endpoint. Results : In the 3 Gy group , pancytopenia was observed , but platelet recovery occured rapidly , returning to normal levels by day 17 post⁃modeling. No deaths occurred during the observation period. At myeloid⁃to⁃erythroid (M/E) ratio , and no significant morphological abnormalities were noted in cells at any devel⁃with hematopoietic cells. In the 4 Gy group , pancytopenia persisted throughout the observation period. The survival rate was 90% . Endpoint analysis showed hypocellular marrow by morphological examination. HE staining indicated minimal fatty infiltration in the bone marrow tissue. In the 5 Gy group , pancytopenia was observed , though erythro⁃cyte counts returned to normal levels by day 24. The survival rate during the observation period was 50% . Endoint analysis revealed vacuolization of marrow particles and reduced hematopoietic cells with predominantly non⁃hematopoietic cells in bone marrow morphology. HE staining demonstrated severe fatty infiltration in the bone mar⁃row tissue , with scarcity of immature cells and hematopoietic precursor cells. Flow cytometry analysis showed a de⁃creased proportion of CD4 + T cells (% ) and an increased proportion of CD8 + T cells (% ) . ELISA confirmed elevated secretion of negative hematopoietic regulators : interferon⁃gamma ( IFN⁃γ) and tumor necrosis factor⁃alpha (TNF⁃α ) . Conclusion Combined administration of varying radiation doses with allogeneic lymphocyte infusion consistently induced peripheral blood cytopenia in mice , characterized by reductions in RBC , WBC , and PLT counts. Integrated analysis of bone marrow morphology , histopathological assessment via HE staining , and immuno logical parameters confirmed that a mouse model of aplastic anemia can be successfully established using 5 Gy X ⁃ ray irradiation coupled with infusion of 2 × 106 allogeneic lymphocytes.

Objective To investigate the clinical characteristics and prognosis of acute myeloid leukemia(AML)patients with PTPN11 gene mutation.Methods Total 115 adult AML patients who underwent initial diagnosis,treatment,and second-generation sequencing(NGS)detecting at hospital were recruited in this study.Clinical da-ta included disease characteristics,treatment efficacy,long-term prognosis,immune cell subpopulations,and leu-kemia stem cells were collected to analyze the clinical characteristics and prognosis of AML patients with PTPN11 gene mutation.Results PTPN11 gene mutation rate in newly diagnosed adult AML was 9.57％,and the mutation site mainly occurred in exon 3 region with all mutation type being point mutation.Compared with PTPN11 wild-type group,PTPN11 gene mutation group had a higher early mortality rate(18.18％vs 4.00％,P=0.048),a lower complete response rate(33.33％vs 67.71％,P=0.039),a higher recurrence rate(83.33％vs 42.31％,P=0.043),a shorter median overall survival time(9 months vs 20 months,P=0.026),a lower proportion of ef-fector T cells[(1.39±0.12)％vs(3.56±0.46)％,P=0.038],and a higher proportion of leukemia stem cells[(13.82±3.66)％vs(3.87±1.40)％,P=0.021].Conclusion PTPN11 gene mutation is a poor prognostic marker for AML.Those patients have a high early mortality rate,low complete remission rate,high recurrence rate,short median overall survival time,a low proportion of effector T cells,and a high proportion of leukemia stem cells.

Objective:To investigate changes and clinical significance of senescent cell proportion and senescence-associated cytokines in peripheral blood of patients with acute myeloid leukemia(AML)in different states.Methods:AML patients who were diagnosed and treated in the Second Hospital of Anhui Medical University were selected as research subjects(17 cases in newly diag-nosed group,16 cases in remission group,and 12 cases in relapse group),morphology of leukemia blasts and proportion of senescent cells in peripheral blood of patients in each group were observed by Regis and senescence specific β-galactosidase(SA-β-gal)staining,levels of senescence-associated cytokines IL-8,IL-1β,IL-6,IL-10 in peripheral blood of each group were detected by cytometric bead array(CBA),and differences and clinical significance among groups were compared.Results:Relapse group peripheral blood smear Regis staining showed that morphology of leukemia blasts increased significantly and proportion of SA-β-gal positive cells was significantly increased(P<0.01);levels of serum senescence-associated cytokines IL-8,IL-1β,IL-6 and IL-10 in relapse group were significantly increased(P<0.05);there was no correlation between serum senescence-associated cytokine levels and proportion of peripheral blood leukemia blasts(P>0.05);relapsed patients with higher IL-8 levels trend to have lower re-induction remission rates and worse overall survival after relapse(P<0.05);multivariate Cox regression analysis showed that IL-8 level was an indepen-dent risk factor for prognosis of relapsed patients(P<0.05).Conclusion:Increased proportion of senescent cells and significantly increased levels of senescence-associated cytokines IL-8,IL-1β,IL-6,IL-10 in peripheral blood of AML patients are closely related to disease recurrence,and relapsed patients with higher IL-8 level had poor prognosis.

Objective To investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia (AML) by targeting paired box 9 (PAX9). Methods The relationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis (GEPIA) database and The Cancer Genome Atlas (TCGA) database. Kasumi-1 and AML5 cells were transfected with empty vector (Vector group) or PAX9 (PAX9 group). The proliferation activity was detected by CCK-8 assay, and cells cycle and apoptosis were detected by flow cytometry. Expressions of cyclin-dependent kinase 2 (CDK2), cyclin B1 (CCNB1), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX) were detected by Western blot analysis. The targeted microRNA (miRNA) by PAX9 was predicted by bioinformatics analysis, and the targeted effect was verified by luciferase reporter assay. The level of PAX9 mRNA was detected by real-time quantitative PCR, and expression of PAX9 protein was detected by Western blot analysis. Kasumi-1 and AML5 cells were transfected with miR-NC (miR-NC group) or miR-181b-5p (miR-181b-5p group). The cells were further transfected with PAX9 (miR-181b-5p combined with PAX9 group) in miR-181b-5p group. The proliferation, cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients, which was correlated with poor prognosis. In Kasumi-1 and AML5 cells, compared with Vector group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in PAX9 group. It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p. Compared with miR-NC group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were increased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were decreased in miR-181b-5p group. Compared with miR-181b-5p group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in miR-181b-5p combined with PAX9 group. Conclusion The miR-181b-5p can promote the proliferation of AML cells and delay apoptosis by inhibiting PAX9.
Humans ; Apoptosis/genetics* ; bcl-2-Associated X Protein ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Leukemia, Myeloid, Acute/pathology* ; Luciferases ; MicroRNAs/metabolism* ; PAX9 Transcription Factor/genetics*

Objective:To investigate the characteristics of gene mutation by using whole exome sequencing (WES) and its relationship with therapeutic efficacy in patients with acute myeloid leukemia (AML).Methods:The data of 30 patients with AML from the Second Hospital of Anhui Medical University between December 2014 and September 2019 were retrospectively analyzed; the result of WES, disease type and classification, genetic prognostic stratification and therapeutic efficacy were summarized. The mutation types and mutation frequency of AML patients stratified by the different clinical characteristics and genetic prognosis were compared.Results:Among 30 AML patients, 26 cases (86.7%) had 1 gene mutation at least. The genes with a mutation frequency more than 10.0% were NRAS, RUNX1, TET2, CEBPA, IDH2 and ASXL1. The function of the mutated genes was involved in signaling pathways, transcription factors, epigenetics, and RNA splicing and other biological functions; in terms of mutation pattern, 19 cases (63.3%) of all AML patients mainly presented combined mutations of many combinations. There were no significant differences in mutation rates among age, gender, disease type, disease classification and genetic prognosis stratification groups (all P > 0.05). Of the 8 fusion gene positive cases, 7 were mutated, the average mutation frequency was 175.0% (14/8). There were 19 mutations in 22 patients with negative fusion gene, the average mutation frequency was 213.6% (47/22), and the difference in mutation frequency between the two groups was statistically significant ( P = 0.001). Of the 14 cases of continuous remission, 11 had mutations, with average mutation frequency of 157.1% (22/14); there were 9 cases of mutations in 10 relapsed patients, and the average mutation frequency was 200.0% (20/10); mutations occurred in 5 patients with primary drug resistance, and the average mutation frequency was 300.0% (15/5); and the difference of mutation frequency among the three groups was statistically significant ( P = 0.009). Conclusions:Through WES analysis, it is found that most AML patients have complex and variable gene mutations; and the higher the gene mutation frequency is, the worse the prognosis of the disease is.

Objective:To explore the safety and efficacy of chimeric antigen receptor T-cell (CAR-T) therapy for relapsed/refractory acute B-cell lymphoblastic leukemia (B-ALL) with T315I mutation.Methods:The clinical data of a patient with relapsed/refractory B-ALL with T315I mutation who underwent CAR-T therapy in the Second Affiliated Hospital of Anhui Medical University was analyzed, and the related literature was reviewed.Results:The patient was a 34-year-old man. He was diagnosed with chronic myelogenous leukemia (CML) in January 2017 and started to take imatinib orally. However, the primary affection transformed to B-ALL 4 months later. Because of the E355G gene mutation, the treatment drug was adjusted to dasatinib, and induction chemotherapy was given at the same time. The sequential consolidation chemotherapy was given for 3 times after complete remission (CR). After half a year of remission, T315I mutation was detected and re-induced chemotherapy was given, but ineffective. The patient was treated with CAR-T 3 days after FC regimen (fludarabine 30 mg/m 2 per day, day 1 to day 3; cyclophosphamide 200 mg/m 2, day 1 to day 3). The number of CD19 CAR-T was 1.0×10 9, 98% activity degree. Grade 1 cytokine-releasing syndrome appeared after infusion, and was resolved after symptomatic treatment. No serious adverse reactions were observed. CR was achieved half-month after CAR-T treatment, and umbilical cord blood transplantation was successfully performed 1 month later. At the last follow-up, the relapse-free survival time of the patient was 396 days. Conclusion:CAR-T therapy may be a new, safe and effective therapy for patients with relapsed/refractory B-ALL with T315I mutation.

Objective: To improve the understanding of high calcium risk in patients with diffuse large B-cell lymphoma (DLBCL) during the chemotherapy. Methods: The diagnosis and treatment of high calcium risk in one patient with DLBCL during the chemotherapy in the Second Affiliated Hospital of Anhui Medical University was retrospectively analyzed, and the relevant literatures were reviewed. Results: A 52-year-old man who was diagnosed with DLBCL (non-specific, non-germinal center source; stage Ⅳ group A; International prognosis index score 4 points, high-risk group) in June 2017. Two times R-CHOP chemotherapy was performed before diagnosis. This patient was admitted to the hospital for the third chemotherapy, and the disease assessment showed that the enlarged lymph nodes were not significantly smaller than before, and the tumor burden was still high. Therefore, the chemotherapy regimen was adjusted to R-GDP regimen. However, on the 8th day after the end of rituximab treatment, the patient had head pain, which might be related to the patient's poor sleep and primary invasion of the primary disease (blood calcium: 2.94 mmol/L). And then the ibuprofen and diuresis treatments were given, but the symptoms were still gradually worsening, and vomiting appeared on the 9th day, systemic fatigue with drowsiness and irritability appeared on the 12th day. Review blood calcium: 5.02 mmol/L. Adequate fluid hydration, diuretic, renal replacement treatments were given, and the level of blood calcium gradually returned to normal. Finally, the patient's symptoms were improved significantly, and he successfully completed R-GDP chemotherapy. Conclusion If a DLBCL patient has symptoms such as headache, lethargy, irritability or even coma during the chemotherapy, it is necessary to alert the possibility of hypercalcemia and to timely improve the relevant examination and make symptomatic treatment.

Objective: To investigate the clinical significance of tumor associated macrophages (TAM) in multiple myeloma (MM) and the relationship with angiogenesis and immunosuppression. Methods: Seventy cases of MM patients diagnosed from August 2015 to June 2017 were enrolled in the study as experimental group, 20 cases of benign hematological diseases (13 with iron deficiency anemia and 7 with megaloblastic anemia) patients as control group. Immunohistochemical method was used to detect the expression of CD163, CD34 and VEGF in bone marrow samples, and flow cytometry was used to detect the proportion of regulatory T cell (Treg cells), ELISA was used to detect the level of IL-10, and the clinical features were analyzed. Results: ①Among the 70 patients, there were 31 males and 39 females with a median age of 65 (50~78) years old. TAM infiltration density, microvascular density (MVD), VEGF expression level, Treg ratio and IL-10 level in bone marrow samples of 70 MM patients were significantly higher than those of benign hematological diseases (P<0.05). ②In the MM group, the above indexes of the patients with disease stabilized (15 cases) were lower than those of the newly diagnosed group (35 cases) and the relapse refractory group (20 cases) (P<0.05), those of relapse refractory group were higher than those of newly diagnosed group (P>0.05). ③Of the 35 newly diagnosed MM patients, 27 completed 4 courses of treatment. In the effective group (15 cases), the TAM infiltration density after treatment was significantly lower than that before treatment, the difference was statistically significant[(20.20±7.66) vs (28.87±11.97), t=2.362, P=0.025]; while in the ineffective group of 12 cases, the difference of the TAM infiltration density before and after treatment was not statistically significant[(42.00±13.76) vs (48.25±13.59), t=1.119, P=0.275]. ④TAM infiltration density in the effective group after bortezomib treatment (21 cases) were lower than those in the non-bortezomib treatment group (18 cases)[(16.52 ±4.26) vs (19.27 ±5.82), t=1.662, P=0.170]. ⑤The TAM infiltration density in MM patients was positively correlated with MVD, VEGF expression level, Treg cell ratio and IL-10 level (P<0.001). Conclusion The infiltration of TAM in the microenvironment of MM, which may promoting angiogenesis and inhibiting immune response, is related to the occurrence, development, therapeutic effect and drug resistance of MM.

Objective To investigate the relationship between red blood cell distribution width (RDW) and prognosis in patients with multiple myeloma (MM).Methods The population that studied consisted of 27 patients with multiple myeloma and 30 healthy controls.The RDW was calculated according to the results of blood routine examination and compared between patients and healthy controls.Then,compared the difference between the two groups of RDW.MM patients were treated with international standard staging (ISS),and the differences of RDW in different stages were analyzed.ISS staging was used to draw the receiver operating curve (receiver operating characteristic curve,ROC curve),then take RDW14.65 ％ as the best cut-off point,the MM patients were divided into low RDW group (RDW=14.65 ％) and high RDW group (RDW ＞14.65％).Overall survival (OS) condition were compared between the above two groups.The impacts of RDW on OS were analyzed by Kaplan-Meier and Log-rank test.Results The average RDW value in experimental and controlled were 15.60 ％ ± 2.35 ％ vs 12.72 ％ ±0.61 ％ separately (t=6.201,P＜0.001),with statistical differences.The average RDW value in low ISS(Ⅰ + Ⅱ stage) and high ISS (Ⅲ stage) were 13.99 ％ ± 1.08％ vs 16.55 ％±2.39％ separately (t=3.800,P=0.001).The median survival time of low RDW and high RDW group was 13 months and 8 months respectively,and the difference was statistically significant (x2=6.481,P =0.011).Conclusion RDW increased in patients with MM,the risk stratification higher prognosis is worse.

Objective To explore the neutrophil to lymphocyte ratio (NLR) and its relationship with the effect of chemotherapy and prognosis in patients with diffuse large B-cell lymphoma (DLBCL).Methods The clinicopathological characteristics and outcome of 51 patients with DLBCL diagnosed by pathological biopsy and immunohistochemistry who received CHOP or R-CHOP regimen were collected and reviewed.According to the median of NLR,the patients were divided into low NLR group (NLR≤2.32) and high NLR group (NLR＞2.32).The prognostic influence of the NLR on overall survival (OS) was studied by Kaplan-Meier method and Log-rank test.To evaluate the independent prognostic relevance of NLR,univariate and multivariate Cox regression models were applied.Results The complete response (CR) rates of the low and high NLR groups were 71.4 ％ (20/28) and 39.1％ (9/23),respectively (P =0.02).The OS in the low NLR group was significantly better than that in the high NLR group (1,2 and 3-year OSs were 96.4 ％,90.4 ％ and 72.3 ％ vs 63.9 ％,52.7 ％ and 42.2 ％,respectively,P =0.009).Univariate and multivariate Cox regression models analysis showed that NLR ＞ 2.32 was an independent prognostic factor (P =0.016).Conclusion An elevated NLR before treatment indicates the poor effect of chemotherapy and prognosis of patients.NLR is an independent prognostic factor for DLBCL.