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Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia in which lymphoid and myeloid markers are co-expressed. Knowledge regarding the genetic features of MPAL is lacking due to its rarity and heterogeneity. Here, we applied an integrated genomic and transcriptomic approach to explore the molecular characteristics of 176 adult patients with MPAL, including 86 patients with T-lymphoid/myeloid MPAL (T/My MPAL-NOS), 42 with Ph+ MPAL, 36 with B-lymphoid/myeloid MPAL (B/My MPAL-NOS), 4 with t(v;11q23), and 8 with MPAL, NOS, rare types. Genetically, T/My MPAL-NOS was similar to B/T MPAL-NOS but differed from Ph+ MPAL and B/My MPAL-NOS. T/My MPAL-NOS exhibited higher CEBPA, DNMT3A, and NOTCH1 mutations. Ph+ MPAL demonstrated higher RUNX1 mutations. B/T MPAL-NOS showed higher NOTCH1 mutations. By integrating next-generation sequencing and RNA sequencing data of 89 MPAL patients, we defined eight molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics. G1 was associated with CEBPA mutations, G2 and G3 with NOTCH1 mutations, G4 with BCL11B rearrangement and FLT3 mutations, G5 and G8 with BCR::ABL1 fusion, G6 with KMT2A rearrangement/KMT2A rearrangement-like features, and G7 with ZNF384 rearrangement/ZNF384 rearrangement-like characteristics. Subsequently, we analyzed single-cell RNA sequencing data from five patients. Groups G1, G2, G3, and G4 exhibited overexpression of hematopoietic stem cell disease-like and common myeloid progenitor disease-like signatures, G5 and G6 had high expression of granulocyte-monocyte progenitor disease-like and monocyte disease-like signatures, and G7 and G8 had common lymphoid progenitor disease-like signatures. Collectively, our findings indicate that integrative genomic and transcriptomic profiling may facilitate more precise diagnosis and develop better treatment options for MPAL.

The low rate of durable response against relapsed and/or refractory multiple myeloma (RRMM) in recent studies indicates that chimeric antigen receptor T‐cell (CART) treatment is yet to be optimized. This study aims to investigate the safety and efficacy of sequential infusion of CD19‐CART and B‐cell maturation antigen (BCMA)‐CARTs for RRMM with a similar 3 + 3 dose escalation combined with a toxicity sentinel design. We enrolled 10 patients, among whom 7 received autologous infusion and 3 received allogeneic infusion. The median follow‐up time was 20 months. The most common grade 3/4 treatment‐emergent toxicities were hematological toxicities. Cytokine‐release syndrome (CRS) adverse reactions were grade 1/2 in 9 out of 10 subjects. No dose‐limited toxicity (DLT) was observed for BCMA‐CAR‐positive T cells ≤5 × 107/kg), while two patients with dose‐levels of 5–6.5 × 107/kg experienced DLTs. The overall response rate was 90% (five partial responses and four stringent complete responses). Three out of four patients with stringent complete responses to autologous CART had progression‐free survival for over 2 years. The three patients with allogeneic CART experienced disease progression within 2 months. These results evidence the sequential infusion's preliminarily tolerability and efficacy in RRMM, and present a simple and safe design applicable for the establishment of multiple CART therapy., We conducted a pilot study to assess the feasibility of the sequential infusion of CD19 and B‐cell maturation antigen (BCMA)‐specific chimeric antigen receptor T‐cell (CART) for relapsed and/or refractory multiple myeloma treatment with a similar 3 + 3 dose escalation design. Translational Significance: The favorable and persistent responses against RRMM were observed in those patients with a combined infusion of autologous CD19‐CART and BCMA‐CART. The emergence of intolerant adverse event (neurotoxicity and sever CRS) was associated with increased dose level of BCMA‐CARTs infusion.

Myeloproliferative neoplasm (MPN) with PCM1-JAK2 rearrangement is a rare disease with poor prognosis and lacks uniform treatment guidelines. Several studies confirmed the efficacy of ruxolitinib in hematological malignancies with PCM1-JAK2 fusion, but the efficacy is variable. Here, we report two patients diagnosed with MPN with PCM1-JAK2 fusion who were treated with ruxolitinib-based regimen, including the first case of ruxolitinib combined with pegylated interferon (Peg-IFN), and we conduct a literature review. We found that ruxolitinib combined with Peg-IFN is an effective treatment option in the case of poor efficacy of ruxolitinib monotherapy.

Patients with relapsed/refractory acute myeloid leukemia (R/R AML) often show resistance to chemotherapy and have dismal outcomes. Therefore, it is urgent to develop new treatment strategies to address this problem. With tremendous achievement of chimeric antigen receptor T cells (CAR-T) therapy against B-cell malignancies, many efforts have been devoted to developing CAR-T therapy for R/R AML but with limited success, in part owing to a lack of specific targets. C-type lectin-like molecule-1 (CLL-1) is highly expressed on AML blasts with no expression on normal hematopoietic stem cells, which makes it an ideal target of immunotherapy for AML. Here, we report 2 R/R AML patients who relapsed after allogeneic stem cell transplantation and failed multiline salvage therapies including anti-CD38 CAR-T therapy, but were successfully treated with PD-1 silenced anti-CLL-1 CAR-T therapy. Both patients achieved molecular complete remission with incomplete hematologic recovery at 28 days of evaluation after CLL-1 CAR-T cell infusion. Cytokine release syndrome in cases 1 and 2 were grade 1 and 2, respectively. At the last follow-up, cases 1 and 2 had maintained continuous remission for 8 and 3 months, respectively. Our results demonstrated that CLL-1 CAR-T cells might be an effective and safe salvage therapy for AML patients with posttransplant relapse.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for acute myeloid leukemia (AML). However, most patients experience relapse after allo-HSCT, with a poor prognosis, and treatment options are limited. The lack of an ideal targetable antigen is a major obstacle for treating patients with relapsed AML. CD38 is known to be expressed on most AML and myeloma cells, and its lack of expression on hematopoietic stem cells (HSCs) renders it a potential therapeutic target for relapsed AML. To investigate the clinical therapeutic efficacy and safety of CD38-targeted chimeric antigen receptor T (CAR-T-38) cells, we enrolled 6 AML patients who experienced relapse post-allo-HSCT (clinicaltrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT04351022","term_id":"NCT04351022"}}NCT04351022). Prior to CAR-T-38 treatment, the blasts in the bone marrow of these patients exhibited a median of 95% (92–99%) CD38 positivity. Four weeks after the initial infusion of CAR-T-38 cells, four of six (66.7%) patients achieved complete remission (CR) or CR with incomplete count recovery (CRi); the median CR or CRi time was 191 (range 117–261) days. The cumulative relapse rate at 6 months was 50%. The median overall survival (OS) and leukemia-free survival (LFS) times were 7.9 and 6.4 months, respectively. One case relapsed 117 days after the first CAR-T-38 cell infusion, with remission achieved after the second CAR-T-38 cell infusion. All six patients experienced clinically manageable side effects. In addition, multiparameter flow cytometry (FCM) revealed that CAR-T-38 cells eliminated CD38 positive blasts without off-target effects on monocytes and lymphocytes. Although this prospective study has a limited number of cases and a relatively short follow-up time, our preliminary data highlight the clinical utility and safety of CAR-T-38 cell therapy in treating relapsed AML post-allo-HSCT.

To define the fusion genes in T/myeloid mixed-phenotype acute leukemia (T/M MPAL), we performed transcriptome sequencing of diagnostic bone marrow samples from 20 adult patients. Our analysis identified a second instance of a recurrent MED14-HOXA9 chimeric gene resulting from the in-frame fusion of exon 23 of MED14 and exon 1 of HOXA9, the first in an adult patient. The MED14-HOXA9 fusion gene was detected in both the diagnostic and relapsed blasts with reverse transcription-polymerase chain reaction and Sanger sequencing. The patient received combined conventional chemotherapy but suffered relapse at 11 months and died of disease progression one year after the initial diagnosis. Our data suggest that MED14-HOXA9 is a cryptic recurrent aberration in T/M MPAL, which might indicate an aggressive clinical course and inferior outcome after conventional chemotherapy. Further studies will be carried out to reveal the effects of the MED14-HOXA9 fusion on the differentiation and proliferation of leukemia stem cells, as well as suitable treatment strategies for this emerging entity.

Philadelphia chromosome-like B-lymphoblastic leukemia (Ph-like ALL) describes a group of genetically heterogeneous, Ph-negative entities with high relapse rates and poor prognoses. A Janus-kinase-2 (JAK2) rearrangement has been reported in approximately 7% of Ph-like ALL patients whose therapeutic responses to JAK inhibitors have been studied in clinical trials. Here, we report a novel STRBP-JAK2 fusion gene in a 21-year-old woman with Ph-like ALL. Although a normal karyotype was observed, a hitherto unreported JAK2 rearrangement was detected cytogenetically. STRBP-JAK2 fusion was identified by RNA sequencing and validated by Sanger sequencing. The Ph-like ALL proved refractory to traditional induction chemotherapy combined with ruxolitinib. The patient consented to infusion of autologous chimeric antigen receptor (CAR) T cells against both CD19 and CD22, which induced morphologic remission. Haplo-identical stem cell transplantation was then performed; however, she suffered relapse at just one month after transplantation. The patient subsequently received donor lymphocyte infusion after which she achieved and maintained a minimal residual disease negative remission. However, she succumbed to grade IV graft-versus-host disease 7 months post-transplant. In conclusion, this report describes a novel STRBP-JAK2 gene fusion in a Ph-like ALL patient with a very aggressive disease course, which proved resistant to chemotherapy combined with ruxolitinib but sensitive to immunotherapy. Our study suggests that CAR T-cell therapy may be a viable option for this type of leukemia.

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To identify whether Pulsatilla saponin A (PsA), an active molecule extracted from Pulsatilla chinensis regel, can induce acute myeloid leukemia (AML) cells differentiate.PsA was isolated from P. chinensis, and its effects of differentiation induction on both AML cell lines and the primary leukemia cells were investigated.Compared with the untreated control, PsA induced the differentiation of U937 cells, K562 cells and HL-60 cells, represented as the increased CD15+ cells in a dose- and time-dependent manner in all the three AML cell lines, after PsA treatment. As the same time, the cell morphology of these AML cells was changed correspondingly; the cytoplasm/nuclei ratio was increased, basophilic cytoplasm was decreased, and eccentric nucleus and granules were also observed. Also, the same effects of differentiation induction by PSA were confirmed in the primary leukemia cells. However, the specific MEK/ERK inhibitor U0126 effectively abrogated the differentiation induced by PsA in vitro.PsA can modify the differentiation activity of AML cells, probably though the MEK/ERK signaling pathway.

The present study aimed to illuminate the role of circulating T follicular helper (TFH) cells in patients diagnosed with chronic immune thrombocytopenia (cITP). Fifty-four patients with cITP and 30 age-matched healthy control subjects were enrolled in the present study. TFH cell frequencies, expression of CD4+ TFH cell-associated cytokines, including interleukin (IL)-2, IL-4, IL-10 and IL-21 and associated regulatory mRNA expression levels including Bcl-6, c-Maf, Blimp-1 and PD-1 pre- and post-treatment with intravenous immunoglobulin and corticosteroids, were detected by flow cytometry, ELISA and reverse transcription-quantitative polymerase chain reaction, respectively. TFH cell frequencies of patients were significantly higher compared with healthy controls pre-treatment (P

Background Increasing evidence suggests that neutrophils play a critical role in tumorigenesis, tumour cell proliferation and metastasis. The prognostic significance of such inflammation-associated markers has been explored in different cancers. Aim To evaluate the prognostic effect of baseline neutrophil counts and nadir neutrophils on advanced gastric cancer (AGC) patients who were treated with two different chemotherapy regimens in our institution. Methods Data were collected retrospectively for 260 AGC patients treated between 1 February 2009 and 31 December 2011. The prognostic effect of baseline neutrophil counts and nadir neutrophils on AGC patients was evaluated. Results Approximately 79% of the patients experienced neutropenia during chemotherapy. The median survival was 369 days for patients with neutrophil counts ≤7.5 × 109/L and 326 days for patients with neutrophil counts >7.5 × 109/L (P < 0.001).The median survival was 340 days for patients with no neutropenia (grade 0), 422 days for patients with mild neutropenia (grade 1–2) and 339 days for patients with severe neutropenia (grade 3–4) (P < 0.001).The adjusted hazard ratios (HR) for mild and severe neutropenia compared with absent neutropenia were 0.572 (P = 0.002) and 1.246 (P = 0.219) respectively. Furthermore, it was suggested that pretreatment baseline neutrophil counts ≤7.5 × 109/L may be an independent predictor (HR = 0.683; P = 0.005). We also observed that other factors were independently associated with worse survival, such as higher performance status, stage IV and the presence of ascites. Conclusion Our findings suggest that baseline neutrophil count and chemotherapy-induced neutropenia can be conveniently available as clinical biomarkers in AGC. Mild myelosuppression in patients with AGC most likely leads to better overall survival, whereas a high baseline neutrophil count may be associated with a worse prognosis.

The Homer protein family, also known as the family of cytoplasmic scaffolding proteins, which include three subtypes (Homer1, Homer2, Homer3). Homer3 can regulate transcription and play a very important role in the differentiation and development for some tissues (e.g. muscle and nervous systems). The current studies showed that Homer3 abnormal expression changes in acute myeloid leukemia (AML). Forced expression of Homer3 in transfected K562 cells inhibited proliferation, influenced the cell cycle profile, affected apoptosis induced by As 2 O 3 through inhibition of Bcl2 expression, and also promoted cell differentiation induced by 12-O-tetra decanoylphorbol-acetate (TPA). These results showed that Homer3 is a novel gene which plays a certain role in the occurrence and development of AML.

To explore the autophagy activity of CD34+ cells in bone marrow of MDS patients and its clinical significance.The activity of autophagy in bone marrow CD34+ cells from 20 MDS patients, 20 non-malignant anemia patients and 5 AML patients admitted in our hospital from October 2012 to March 2014 was detected by flow cytometry (FCM).The autophagy activity in low risk MDS patients and non-malignant anemia patients were both significantly higher than that in both high risk MDS and AML patients (P0.05), and more interestingly, the autophagy activity in MDS negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r=-0.877) .The autophagy activity CD34+ cells in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores, indicating that the decrease of autophagy activity maybe accelerate the genesis and development of MDS and relate with the prognosis of MDS patients.

To investigate the alteration of microparticles (MP) in the recipients following hematopoietic stem cell transplantation (HSCT) and its significance, and to search the early diagnostic indicators of thrombotic complications after transplantation.According to the occurrence of transplantation-associated complications, 94 allo-HSCT patients were divided into 4 groups: thrombotic group (VOD n = 7, TMA n = 2), acute graft-versus-host disease (aGVHD) group (n = 27), infection group (n = 41) and non-complication group (n = 17). Alterations of serum concentration of tissue factor positive microparticles (TF(+) MP) and endothelial microparticles (EMP) were analyzed by flow cytometry during the process of conditioning treatment and the early stage after transplantation. The relation of these 2 kinds of MP with complications was analysed.(1) The levels of TF(+) MP and EMP of patients undogoing allo-HSCT before conditioning treatment were obviously higher than those in normal controls, and showed some elevation during different times, but there was no significant statistical difference. Although the levels of TF(+) MP and EMP at the end of conditioning treatment were some higher than those before conditioning treatment, but there was no statistical difference between them. (2)The levels of TF(+) MP and EMP in thrombotic group were obviously higher than those in aGVHD group and infection group (P0.05). (3)The levels of TF(+) MP and EMP in thrombotic group at different times were significant differences from those in other groups (P0.05), and the levels of TF(+) MP and EMP were no significant difference from those in non-complication group.The increase of the TF(+) MP and EMP levels may be associated with occurrence of thrombosis after transplantation, indicating occurrence of the thrombotic complications, like hepatic vein occulusive disease (HVOD). The dynamically monitoring levels of TF(+) MP and EMP contributes to early discovery of thrombotic complications.

To summarize the clinical characteristics as well as diagnosis and treatment in 1 case of acute myeloid leukemia(AML) with coexpression of Ph and inv(16).A series of clinical tests, the cellular morphological, immunological, cytogenetic and molecular biological examinations of leukemia cells were performed.The clinical characteristics of this patient were very common. The cellular morphology is similar to the AML with inv(16). The leukemia cells were stained positively for CD13, CD33, CD34, CD117 and HLA-DR. Karyotypic analysis showed a complex chromosome abnormality including inv(16) and Ph, and the FISH analysis showed that the percentage of rearrangement of CBFβ allele was over that of the BCR-ABL fusion signals. The obvious adverse events did not occur in this patient within 3 years.Ph as secondary aberration of inv(16) rarely occures in primary AML cases, and so far there have not been the clear criteria of diagnosis and treatment. The cytogenetic and molecular biology could provide the basis for diagnosis. Moreover, autologous hematopoietic stem cell transplantation combined with imatinib probably is one of the effective treatment methods.

Platelet thrombus formation is a major contributor to various cardiovascular diseases caused by vascular occlusion. Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a key role in platelet aggregation and hence the formation of thrombi. In the present study, the genes encoding the light- and heavy-chain variable regions (V(H) and V(L)) of SZ-21, which is a monoclonal antibody (MoAb) against GPIIIa integrin have been cloned by RT-PCR from the SZ-21 hybridoma. The genes of V(H) and V(L) were attached to the oligonucleotide of the linker peptide and single-chain antibody fragment (ScFv) was constructed. The ScFv was ligated into phage display vector pHEN1, and the phagemid pHEN1-21ScFv was constructed. The high-affinity phage display technology was used to retain the SZ-21ScFv binding activity to platelets in great effort. After four rounds of enrichment, the screening clone of SZ-21ScFv gene with good reactivity to platelets was ligated into highly expressed vector pET20b and expressed in Escherichia coli BL21(DE3)PlysS for the fusion protein. Recombinant ScFv fragment was produced mostly in the form of inclusion bodies, with its yield up to 21% of the total amount of bacteria protein. The ScFv fragment with the similar binding activity to platelets as MoAb SZ-21 was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot. ADP-induced platelet aggregation can be inhibited by ScFv fragment in a dose-dependent manner and the maximal inhibition rate was obtained at a concentration of 750 nM. In addition, the ScFv fragment has ability to inhibit the binding of fibrinogen to platelets and react with endothelial cells. In this study, we have successfully produced the SZ-21ScFv, which retained the binding affinity to platelets and antithrombotic ability of their murine counterparts.

To evaluate the evolution of paroxysmal nocturnal hemoglobinuria (PNH) clone and its clinical significance before and after immunosuppressive therapy (IST) in patients with aplastic anemia (AA).A total of 186 patients diagnosed as AA were enrolled in this study. Among them, 55 patients were diagnosed as severe AA (SAA) and treated with cyclosporine (CsA) plus anti-thymocyte globulin (ATG), 131 were diagnosed as non SAA (NSAA) and treated with CsA alone. All patients were screened for PNH clone by flow cytometry before treatment and followed up for 18-76 months, with a median time of 22 months.Positive PNH clones were detected in 10 SAA (18.9%) patients, significantly more than that of NSAA group [9 patients (7.4%), t = 5.041, P = 0.025]. The proportions of PNH clones in SAA group at 6, 12, 24 and24 months were 13.38%, 14.88%, 20.00% and 18.85%, respectively, also significantly higher than those of NSAA patients (5.67%, 5.31%, 5.47% and 9.08%, all P values0.05). Clinical response rates were comparable in both ATG+CsA or CsA alone groups no matter PNH clone was positive or negative.PNH clone are detectable in AA patients either treated with ATG plus CsA or CsA alone, and more significant by ATG plus CsA. Whether PNH clone occurred before or after IST does not affect the therapeutic efficacy.

The purpose of this study was to summary the clinical and laboratorial features in 15 adult cases of mixed phenotypic acute leukemia with Ph chromosome and/or BCR-ABL fusion gene positive (Ph(+)MPAL), 15 adult patients with Ph(+)MPAL were defined by WHO-2008 classification. The clinical characteristics, results of morphology, immunology, cytogenetics and molecular genetic detections and results of follow-up in 15 adult patients with Ph(+)MPAL were analyzed retrospectively. The results showed that 15 patients among 87 cases of MPAL demonstrated Ph(+)MPAL (17.2%; 15/87) (7 males and 8 females), their median age was 51 (range 16-81) year old and median WBC count at diagnosis was 69 (12.7-921)×10(9)/L. Based on FAB criteria, these patients showed different morphologic types, including AML (13.3%; 2/15), ALL (40.0%; 6/15), HAL (46.7%; 7/15). Immunologic analysis indicated that 15 cases of Ph(-)MPAL were all classified as B-lymphoid +myeloid mixed immunophenotype. Except one patient, all expressed CD34 antigen on the surface of leukemia cells with 64.3% strong positive, only Ph (53.3%; 8/15), Ph with additional chromosomal abnormalities (33.3%; 5/15) and normal karyotype (13.3%; 2/15) were cytogenetically identified. BCR-ABL fusion gene transcript positive were detected by multiplex reverse transcription PCR in all cases, with e1a2 subtype (p190) (40.0%; 6/15) and b2a2 or b3a2 (p210) subtype (60.0%; 9/15). Four out of 7 (57.1%) patients were found to have IKZF1 gene deletion, without other common gene mutations. Seven out of 10 cases (70.0%) achieved complete remission (CR) after one cycle of induction chemotherapy. In the induction stage, CR rate seemed higher when tyrosine kinase inhibitors (TKI) were added to chemotherapy (83.3%:50.0%; P = 0.206). Overall survival (OS) in 4 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) was longer than that in 4 patients received chemotherapy alone (P = 0.004). It is concluded that Ph(+)MPAL mainly is expressed as B+My phenotype. The majority of patients is older and has CD34 overexpression. In the aspect of molecular genetics, the Ph(+)MPAL is similar to other acute leukemia with Ph chromosome. Ph(+)MPAL is a subtype of acute leukemia with poor prognosis. WBC count at diagnosis is an independent prognostic factor. The combination of TKI and allo-HSCT can improve their long-term survival, which needs to be confirmed through carrying out a prospective and multicenter clinical trial for newly diagnosed Ph(+)MPAL.

To evaluate the possible mechanism of transcription factors B cell lymphoma 6 (Bcl-6) , forkhead/winged helix transcription factor 3 (Foxp3) and retinoic acid related orphan receptor (RORγt) in CD4(+) T cells for immuno-related hematocytopenia (IRH).CD4(+) T cells were harvested from 40 IRH patients, 38 aplastic anemia subjects and 25 normal controls and separated by magnetic activated cell sorting (MACS). Then the expressions of transcription factors of Foxp3, RORγ and Bcl-6 in CD4(+) T cells were measured by real time fluorescent quantitative-polymerase chain reaction (QRT-PCR).Auto-antibody was detected on CD34(+) cells (67.5% (27/40) ), CD15(+) cells (65.0% (26/40)), GlyA(+) cells (75.0% (30/40) ), auto-antibody involving three, two or one myeloid cell were detected in 27.5% (11/40), 52.5% (21/40), 20.0% (8/40) of IRH patients. Compensatory increase of Foxp3 mRNA was found in IRH (0.124 (0.073-0.198) vs 0.071 (0.046-0.118), P0.05). The expression of Bcl-6 was higher (2.243 (0.854-4.544) vs 1.211 (0.131-2.816), P0.05). Compared to aplastic anemia, the expression of RORγt was lower in IRH (0.133 (0.068-0.189) vs 0.290 (0.138-0.480), P0.01) and the ratio of Treg/Th17 shifted to Th17 in patients with aplastic anemia (Foxp3/RORγt ratio,0.500 (0.240-0.795) vs 0.975 (0.483-1.416), P0.01).As one kind of bone marrow failures caused by autoantibody to bone marrow cells, IRH may occur due to a high expression of Bcl-6 in CD4(+) T cells, its immunopathogenesis is different from that of aplastic anemia.

The goal of this study is to develop a flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies commonly present in bleeding patients with immune thrombocytopenic purpura (ITP).Polystyrene microbeads coated with antibodies against human platelet glycoproteins (GPs) IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51) were incubated with platelet lysate from 50 ITP patients and 86 controls. The platelet antigen-autoantibody complexes were detected by flow cytometry using an FITC-labeled antibody. The results were compared with that of a monoclonal antibody immobilization of platelet antigen (MAIPA) assay.By FCIA, platelet autoantibodies against GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in ITP patients. Mean fluorescent intensity values with antibodies SZ1, SZ2, SZ21, SZ22 and SZ51 were all higher in ITP patients than controls (p values0.01). In ROC analysis, values of the area under the curve were 0.89, 0.82, 0.93, 0.94 and 0.95, respectively. In ITP diagnosis, the FCIA assay with these five antibodies had better sensitivity and accuracy than the MAIPA assay (96% vs. 44% in sensitivity; 80.9% vs. 64.7% in accuracy, p0.01).FCIA assays with multiple antibodies against platelet GPs may be used to improve the diagnosis of ITP in hospitals.

Objective of this study was to detect the level of tissue factor-positive microparticles (TF(+)MP) by flow cytometry (FCM) and to analyze its clinical significance in the haemostatic disorder. TF(+) MP was detected by FCM using antibody CD142-PE in 25 cases of acute promyelocytic leukemia (APL), 20 cases of hemostatic diseases and 20 healthy adults as controls. The differences of TF(+) MP between various groups were determined. The results showed that the level of TF(+) MP in the patients with thrombotic complications was significantly higher than that in the healthy adults (P0.05). The TF(+) MP level was higher in the patient with APL than that in the healthy adults, especially in course before therapy (P0.01), but the difference was not statistically significant in the patient with APL after therapy and the healthy adults. Among these patient with APL, the level of TF(+) MP in the 18 patients who complicated with disseminated intravascular coagulation (DIC) was also higher than that in the healthy adults (P0.05), but the level of TF(+) MP in the other 7 patients who did not complicate with DIC was similar before and after treatment. It is concluded that the method of TF(+) MP detection by FCM is feasible and simple, it is useful for the diagnosis of thrombotic disorder, and helps evaluation for the prognosis of APL patient.

To explore the relationship between minimal residual disease (MRD) and the outcome of patients with high-risk acute leukemia (AL) undergoing allogeneic hematopoietic stem cell transplantation (HSCT).By 4/5-color multi-parameter flow cytometry (MFC, CD45/SSC gating) for detecting MRD at pre-(day-30) and post-transplant (day +30, +60, +100, 6 months, 9 months and 12 months), the investigators retrospectively analyzed the MRD levels and the prognosis of 90 high-risk patients. According to the MRD cutoff value of 0.1%, the low-level and high-level groups were defined. In the high-level group, the patients were divided into two sub groups according to the subsequent treatment (intervention therapy group and non-intervention therapy group).MRD pre-transplant had no predictive value for the clinical outcome. The patients with high levels of MRD post-transplant (+60 d and +100 d) showed higher relapse rates than those of the low-level group. In addition, regarding MRD +100 d post-transplant, differences were significant among 3 groups (high-level MRD and intervention therapy group, high-level MRD and non-intervention therapy group and low-level MRD group) including 1-year relapse-free survival (RFS) (100% vs 60.87% vs 91.30%, P0.05) and 3-year RFS (85.71% vs 44.72% vs 68.48%, P0.05). The median time from first high level MRD detected to clinical relapse was 2.5 (1 - 26) months. In the high level MRD group (+100 d post-transplant), 7 of 30 patients received intervention therapy without relapse. However another 23 patients had no intervention treatment and 11 of them relapsed latter (P0.05).The MFC-based quantification of MRD post-transplant reveals important prognostic information in patients with high-risk AL. MRD check point at day +100 (cutoff: 0.1%) may discriminate different risk populations. Those patients with MRD levels ≥ 0.1% should receive early intervention at an early stage and a low tumor burden so as to reduce the relapse rate and boost survival.

This study was aimed to investigate the immunophenotypic characteristics of 109 cases of B-cell chronic lymphoid leukemia (B-CLL) so as to provide evidences for the diagnosis and therapy of B-CLL, and for the detection of the minimal residual disease and its prognosis. Immunophenotyping was performed in 109 patients of B-CLL by two/three color multiparameter flow cytometry analysis using a panel of monoclonal antibodies. The results showed that in 109 cases of B-CLL, all cases expressed CD19, the positive ratios of other B lineage antigen such as CD20, CD22 and CD23 were 95.40%, 94.50%, 86.20% respectively. None of the B-CLL cases expressed CD10. The expression ratio of FMC-7 and CD38 in 105 cases of B-CLL were 28.60% and 36.20%. Among the B-CLL cases the CD5(+) cells amounted to 86.23%, CD5(-) cells amounted to 13.76%, ZAP-70 protein was expressed in 12 out of 50 patients. It is concluded that immunophenotypic data are very useful for the diagnosis and detection of minimal residual disease of B-CLL, and the relationship between the immunophenotypic characteristics and the prognosis of B-CLL needs further study.

To investigate the clinical and biological characteristics and prognosis of mixed phenotype acute leukemia (MPAL).Thirty two patients were diagnosed as MPAL by bone marrow examination, immunophenotyping, cytogenetic and molecular assay and were treated with combined chemotherapy regimens for both acute lymphoblastic and acute myeloid leukemia. Two cases were received allogeneic hematopoietic stem cell transplantation (allo-HSCT).(1) The incidence of MPAL in acute leukemias was 2.6%. There were 16 cases (50.0%) of mixed myeloid and B-lymphoid (M/B), 14(43.8%) myeloid and T-lymphoid (M/T), one each (3.1%) of trilineage (M/B/T) and B- and T-lymphoid (B/T) phenotype. (2) The positive rates of CD34 and HLA-DR were 87.5% and 62.5%, respectively. (3) Abnormal karyotypes were detected in 70.0% of 30 MPAL patients, which were structural and numerical abnormalities including t(9;22), 11q23 and complex karyotypes. (4) The total complete remission (CR) rate was 75.0% and the overall survival (OS) and disease-free survival (DFS) at 2 years were 14.8% and 14.2% respectively. The CR rates for M/B and M/T cases were 75.0% and 71.4% respectively. No statistical difference was observed in OS and DFS between M/B and M/T cases.MPAL is a rare type of acute leukemia with a high heterogeneity. The unfavorable indicators of MPAL may be factors such as abnormal karyotypes, high expression of CD34 and extramedullary infiltration. Combined regimens and more intensive therapy including allo-HSCT might contribute to improving survival.

To investigate the effect of thalidomide on Annexin II (AnxA2) gene regulation in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide.RPMI8226 and HMEC-1 cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry (FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide.AnxA2 mRNA level in RPMI8226 cells treated with thalidomide at 12.5 microg/ml, 25.0 microg/ml and 50.0 microg/ml was decreased compared with the control group (0.60+/-0.15, 0.33+/-0.14, 0.42+/-0.16, vs 1.07+/-0.16, respectively, P0.05) and did so in HMEC-1 cells (0.21+/-0.20, 0.08+/-0.08, 0.17+/-0.16 vs 1.16+/-0.24, respectively, P0.05). The AnxA2 protein level in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39+/-0.32, 2.82+/-0.28, 3.21+/-0.23 vs 5.53+/-0.32, respectively, P0.05) and that did so in HMEC-1 cells (0.72+/-0.11, 0.64+/-0.08, 0.67+/-0.08 vs 1.40+/-0.15, respectively, P0.05).Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalidomide in multiple myeloma patients.

To investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells.A synthesized siRNA duplex targeting to AnxA2 was transfected into Jurkat cells. Transfection efficiency was analyzed by real-time PCR and flow cytometry. MTT assay for cell proliferation and transwell plates for invasive potential were performed.Compared with the negative controls, the cell proliferation inhibitory rate of the AnxA2 siRNA transfected Jurkat cells was significantly increased at 24 h, 48 h and 72 h [(17.4 +/- 2.3)%, (22.4 +/- 3.8)%, (37.6 +/- 1.5)% vs (-1.3 +/- 5.1)%, (-5.5 +/- 4.4)%, (-10.8 +/- 5.5)%, respectively, P0.05]. The cell invasive potential of the transfected Jurkat cells was inhibited remarkably at 48 h (11.3 +/- 4.2 vs 54.3 +/- 8.7, P0.01).AnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.

To explore the expression of CD34 in patients with acute promyelocytic leukemia (APL) and investigate the clinical and laboratory features of CD34(+) APL patients.262 APL patients diagnosed by chromosome analysis and/or fusion gene examination in the last five years were retrospectively analyzed in this study. To survey the expression of CD34 in those patients, all the cases were divided into two groups (CD34(+) APL vs. CD34(-) APL). The clinical features including age, gender, abnormal values of the peripheral hemogram before treatment, the complete remission (CR) rate and the incidence of DIC and laboratory data such as the results of morphology, immunology, cytogenetics and molecular biology (MICM) between those two groups were compared.Of the 262 APL patients, 38 (14.5%) cases were positive for CD34 expression. There were no statistically significant differences between CD34(+) APL and CD34(-) APL groups in gender and age (P0.05). Before treatment, the median level of WBC in CD34(+) APL was 25.92 x 10(9)/L, which was significantly higher than that of CD34(-) APL (5.3 x 10(9)/L, P0.05). CD34(+) APL by morphology classification were mostly of the subtypes M3b and M3v (65.8%), while these subtypes in CD34(-) APL (40.3%) were significantly less (P0.01). There were no statistically significant differences between the two groups compared in respect of complete remission (CR) rate and the incidence of DIC (P0.05). The expression level of CD34 in APL had correlation to the expression level of CD2, CD7 and CD117; the latter three phenotypes in CD34(+) APL were significantly higher than those in CD34(-) APL (P0.01). No significant difference was found between those two groups by chromosome analysis, but there was more PML-RAR-alpha transcript short form in CD34(+) APL than that in CD34(-) APL (P0.05).CD34(+) acute promyelocytic leukemia is a unique subtype of APL with different biological characteristics.

To investigate the clinical and laboratory features of acute promyelocytic leukemia (APL).513 APL patients in the last two decades were retrospectively analyzed in this research. We investigated the clinical features including age, sex, abnormality of peripheral hemogram before treatment, therapeutic effect and follow-up and laboratory data such as morphology, immunology, cytogenetics and molecular biology (MICM).The median age of the APL patients was 33 years old and the ratio of male and female was 1.21:1. Before treatment, the median level of WBC was 4.3 x 10(9)/L and the detection rate of abnormal promyelocyte on blood film was 85.8%; with immunophenotypic detection, the expression levels of CD117, CD34, HLA-DR, CD7, CD14 and CD19 in APL were found to be lower and the expression levels of CD2, CD33 and MPO higher than those in other subtypes of acute myelocytic leukemia (AML) (both P0.01). Specific abnormal chromosome t (15;17) was detected in 91.7% of the patients, of whom 75.9% had standard translocation of t (15;17), being the most common one and 15.8% of the patients had t (15;17) with additional abnormal chromosome. There was only 7.5% of the patients with normal karyotype. However, the presence of both simple translocation and complex translocation was seldom seen. With molecular biological detection, PML/RARalpha fusion gene positive rate was 99.6%. In a relatively long clinical follow-up, we found that the complete remission (CR) rate in APL patients was 84.7%, incidence of DIC was 13.4% and five-year survival rate was 30.7%. The median count of WBC in CR group was lower than that non-remission group (P0.01). There were no significant differences on expressions of CD34 and CD2 and changes of cytogenetics between the two groups (P0.05).Comprehensive evaluation of MICM could be of important significance in the diagnosis and prognosis judgment for APL patients. The CR rate in these patients with high WBC count was considerable low.

To report a case of pure erythroid leukemia.The clinical features, treatment and prognosis of a rare case of pure erythroid leukemia were reported, and the related literature was reviewed.The pure erythroid leukemia patient was diagnosed by 90.4% pronormoblasts in bone marrow, 99.5% for erythroid antigen CD71, 67.4% for glycophorin A were detected, while no differentiation antigen of myeloid, lymphoid and megakaryocyte lineages were observed. HAG (homoharringtonine + Cytarabine and G-CSF) regimen were administered with no effect. The patient developed multiple organ failure and died soon.Pure erythroid leukemia has a fulminant clinical course with poor response to chemotherapy and worse prognosis.

To explore the characteristics of morphology, immunophenotype and cytogenetics (MIC) of myeloid surface antigen-expressing acute lymphoblastic leukemia (My+ ALL).One hundred and twenty untreated acute lymphoblastic leukemia (ALL) patients were diagnosed by standard bone marrow smear morphologic analysis and peroxidase staining. Flow cytometry and myeloid monoclonal antibodies (McAb) were used to analyze immunophenotype. Chromosome karyotypes were analyzed by R-band technique.Of 120 cases, 66 (55%) were My+ ALL, including 50 cases of My+ B-ALL (56.8% of B-ALL ), 14 cases of My T-ALL (50% of T-ALL) and 2 cases of My+ T and B-ALL (50% of T and BALL). Of 66 My+ ALL, 10 cases (15.1%) were misdiagnosed as acute non-lymphoblastic leukemia (ANLL), the other 54 My- ALL cases were correctly diagnosed. The inconsistent rate between morphological and immunophenotype classifications was higher in My+ ALL than in My- ALL , and there were more atypical morphology cases in My+ ALL than in My- ALL (P0.01). In My+ ALL cases 95.5% expressed CD13, 81.8% CD33, 77.3% CD13 and CD33 simultaneously, and 1.5% CD117, but none CD14, CD15 and MPO. CD34 expression rate in My+ ALL cases was significantly higher than that in My- ALL (P0.01 ). Cytogenetic abnormalities rates in My+ ALL and My- ALL were 72.3% and 66.7% (P0.05) respectively. t(9;22) and t(9;22) plus other cytogenetic abnormalities were detected more frequently in My+ LL cases than in My- B-ALL (P0. 01), and not in My+ T-ALL and My- T-ALL cases. The complete remission (CR) rates was 83.9% in My+ ALL and 79% in My- ALL(P0.05).My+ ALL had a specific characteristics in morphology, immunophenotype and cytogenetics. Some cases have a myeloid morphologic appearance and might be misdiagnosed as acute myeloid leukemia (AML). My+ ALL have a higher CD34 expression rate than My- ALL. t(9;22) abnormality was more frequently observed in My B-ALL than in My- B-ALL. There was no significant difference in CR rate between My+ ALL and My- ALL.

The study was aimed to investigate the immunophenotypic and cytogenetic features of chronic lymphocytic leukemia (CLL) in order to provide an evidence for diagnosis and therapy. Immunophenotypic analysis was performed by using a panel of monoclonal antibodies and three-color immunofluorescence staining methods of flow cytometry in 51 patients with CLL, and the cytogenetic features were analyzed by R-banding technique. The results indicated that among 51 CLL cases, the positive rate of CD19 and CD23 was 96.1%, followed by CD15 (94.1%), CD20 (82.4%) and CD22 (78.4%). The positive rate of CD38 was 23.5%. Forty-six patients expressed both CD5 and CD19. Seven main clonal chromosomal abnormalities were detected by conventional cytogenetics (CC) in eighteen cases (35.3%), with three cases of +12, two cases of 13q(-), other chromosomal abnormalities included +14, 6q(-), t (11; 14), t (14; 18) and t (2; 7). Expression of the antigens had no relationship with chromosomal abnormalities. It is concluded that typical CLL express CD5, CD19 and CD23, and the positive rate detected by CC in CLL is low. Immunophenotyping in combination with cytogenetic technique plays an important role in the diagnosis and prognosis of CLL.

To explore the diagnostic value of intracellular antibody combination in acute leukemia (AL) expressing cross-lineage cell-surface antigens.Flow cytometric immunophenotyping using intracellular antibody combination (cMPO/cCD79alpha/cCD3/CD45) was performed additionally in 60 patients who expressed cross-lineage antigens from 269 previously untreated adult AL.Fifty-four of 269 previously untreated adult AL patients who expressed only one kind of intracellular antigen were diagnosed as cross-lineage AL, the percentage of cross-lineage AL in T cell acute lymphoblastic leukemia (T-ALL), B-ALL and acute myeloid leukemia (AML) was 28.6%, 43.6% and 13.4%, respectively. The positive rate of CD7, CD19, CD5 and CD20 in cross-lineage AML was 65.4%, 15.4%, 11.5%, and 7.7%, respectively. The positive rate of CD13, CD33 and CD15 in cross-lineage ALL was 89.3%, 21.4% and 3.6%, respectively. Six (2.3%) patients expressed two-lineage intracellular antigens were diagnosed as biphenotypic AL: 2 of T/B type and 4 B/M (B/myeloid) type.Intracellular antibodies possess lineage specificity and four-color combination flow cytometric immunophenotyping can provide fast and multi-parameter data. To ensure accuracy of the results, CD45/SSC gating and normal cells as internal reference should be used in the immunophenotyping of abnormal cells.

To report abnormal expression of cCD79a/cCD22 in four cases of acute myeloid leukemia (AML) with t (8;21).The characteristics of morphology, immunophenotype, chromosome karyotype (MIC) and clinical manifestations of 4 AML patients with t (8;21) expressing cCD79a/cCD22 were analyzed.The features of the 4 patients were: (1) no difference in gender; (2) young age; (3) exmedullary infiltration may be present; (4) normal number of white blood cells in peripheral blood; (5) morphology showed acute myeloid leukemia with high percentage of blast cells; (6) B-lymphoid and myeloid immunophenotype, and high expression of CD34; (7) frequent depletion of Y chromosome and complex changes of chromosomes; (8) positive for AML1/ETO fusion gene; (9) response well to chemotherapy regimen which simultaneously treated myeloid and lymphocytic leukemia.Abnormal expression of cCD79a/cCD22 in AML with t (8;21) (q22;q22) suggested that this kind of leukemia might be related with abnormal expression gene of B cell.

Measurement of platelet-associated imunoglobulin (PAIg) has frequently been applied for the diagnosis of idiopathic thrombocytopenic purpura (ITP) and other immune thrombocytopenias. In the present study, a flow cytometry (FCM) analysis has been used to detect and characterize PAIg in 47 patients with ITP and Evans' syndrome, 13 patients with non-immune thrombocytopenia, 10 patients with autoimmune hemolytic anemia (AIHA) whose platelet counts were in normal range, and 31 healthy volunteers. With FCM measurement, mean fluorescence intensity (MFI) of platelets from patients with ITP and Evans' syndrome (2.26 +/- 2.29) was significantly higher than those from non-immune thrombocytopenia (0.33 +/- 0.39), AIHA (0.17 +/- 0.07) and control subjects (0.25 +/- 0.15) (P0.01). Meanwhile, the percentage of positive platelets of patients with ITP and Evans' syndrome [(44.1 +/- 29.0)%] was also higher than those of non-immune thrombocytopenia [(17.5 +/- 9.4)%], AIHA [(10.7 +/- 7.5)%] and control subjects [(16.6 +/- 8.4)%] (P0.01). In addition, some peak shape abnormality appeared (double peaks and peak tail) in the histogram of fluorescence intensity (log) of 11 patients (23.4%) with ITP and Evans' syndrome either alone or accompanied with quantitative alteration of MFI and/or positive platelet percentage. In seven cases, the peak shape abnormality was the unique characteristic that could be detected and have never been seen in normal platelets. This phenotypic alteration perhaps reflects the existence of different platelet populations and could be of diagnostic value. Totally, the positive result of FCM measurement in patients with ITP and Evans' syndrome was 87.2%, slightly higher than 83.0% positive rate with ELISA method, without statistical difference. The correspondent rate of the results of these two analytical settings was 85.1%. This study shows that FCM assay is a rapid and sensitive method for the measurement of PAIg and seems to be suitable as a novel routine diagnostic technique of immune thrombocytopenia.

Drug resistance is a major factor in chemotherapeutic failure of leukemia. Multidrug resistant cell lines are the good models for investigating the mechanisms and reversal of acquired drug resistance. This study was designed to explore the multidrug resistance (MDR) mechanisms in cell line HL-60/VCR.Flow cytometry and a panel of antibodies were used to analyze the expression of MDR proteins (P-gp, MRP, LRP, BCRP, GST-pi) and apoptosis-modulating proteins (bcl-2, bcl-x, bax, bad) in MDR cell line HL-60/VCR and drug sensitive cell line HL-60.The expression levels of MDR proteins (P-gp, MRP, BCRP, GST-pi) were (18.62, 1.19, 1.50, 1.32-flod) higher in HL-60/VCR than in HL-60, while the expression of LRP level was similar. The levels of apoptosis-modulating proteins(bcl-2, bcl-x, bad) were (2.48, 1.25, 1.08-fold) higher in HL-60/VCR than in HL-60, while the pro-apoptosis protein bax contrarily decreased in HL-60/VCR.Various MDR mechanisms were involved in multi-drug resistance HL-60/VCR cell line, which including increasing expression of drug-resistance protein (P-gp, MRP, BCRP, and GST-pi); the apoptosis-modulating proteins (bcl-2, bcl-x, bax, and bad) might take part in the mechanism of drug resistance.

Background CD4+CD25+ regulatory T cell (Treg), the most significant subset of regulatory T cells, has been found to play an important role in suppressing allogenetic immune response and controlling GVHD recently. The present single-center study monitored the ratio of CD4+CD25high T cells and the expression of FOXP3 gene in peripheral blood post-transplant to assess the reconstitution of CD4+CD25high Treg early after allogenetic stem cell transplantation and its correlations with aGVHD. Methods: 22 patients undergoing allo-HSCT were enrolled. To detect the CD4+CD25+T cells and the CD4+CD25high T cells subpopulations ratio in CD4+ T lymphocytes(CD4+CD25+/CD4+ ACD4+CD25high/CD4+) Athe CD4+CD25high T cells subpopulations ratio in CD4+CD25+ T cells(CD4+CD25high/CD4+ CD25+) Aand the expression of FOXP3 mRNA in the donor graft and periperal blood frequently after transplantation with the flow cytometry and real-time PCR assays. Results: \|[Dagger]\|@CD4 +CD25high/CD4+ and CD4+CD25high/CD4+CD25+ ratios in the donor graft were significantly lower in the aGVHD group(P Conclusion: \|[Dagger]\|@The early activation and proliferation of the CD4+CD25high regulatory T cells is critical for the maintainance of immune hemostasis and the control of aGVHD.‡AThe occurrence of aGVHD is related with lower ratios of CD4+CD25high regulatory T cells in CD4+T cells and activated CD4+ effector T cells.‡BaGVHD may affect the quantities of CD4+CD25high regulatory T cells and the expression of FOXP3.