Integrative genomic and transcriptomic profiling reveals distinct molecular subsets in adult mixed phenotype acute leukemia.

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.

Successful application of PD-1 knockdown CLL-1 CAR-T therapy in two AML patients with post-transplant relapse and failure of anti-CD38 CAR-T cell treatment.

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.

A Recurrent Cryptic MED14-HOXA9 Rearrangement in an Adult Patient With Mixed-Phenotype Acute Leukemia, T/myeloid, NOS.

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.

Rapid Molecular Response to Dasatinib in a Pediatric Relapsed Acute Lymphoblastic Leukemia With NCOR1-LYN Fusion.

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of treatment failure and poor outcome. Activation of ABL/Src family kinases is found in ~10% of Ph-like ALL, which can be therapeutically targeted by tyrosine kinase inhibitors. LYN is a member of the ABL/Src-tyrosine kinase family. Somatic LYN rearrangements are found in 5 cases of hematopoietic malignancies so far, although none of them were treated with tyrosine kinase inhibitors. Case presentation: A 6-year-old boy with relapsed B-ALL had no response to reinduction chemotherapy. He was then treated with the ABL1 tyrosine kinase inhibitor dasatinib and achieved complete remission within 2 weeks. Haploidentical allogenic stem cell transplantation (allo-HSCT) was subsequently performed and maintenance therapy with dasatinib initiated 8 weeks post-transplantation. He has been in minimal residual disease negative remission for 10 months after allo-HSCT. Result: His bone marrow karyotype showed a balanced translocation between chromosomes 8 and 17, leading to a NCOR1-LYN fusion gene confirmed with sequencing. Conclusion: Although LYN overexpression is described in many AML and B-ALL patients, intragenic LYN rearrangement is a rare event. For the first time, we present evidence that dasatinib is effective in treating a pediatric B-ALL with NCOR-LYN fusion.

Identification of STRBP as a Novel JAK2 Fusion Partner Gene in a Young Adult With Philadelphia Chromosome-Like B-Lymphoblastic Leukemia.

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.

[Clinical Features and Prognosis of 188 Patients with Acute Myeloid Leukemia-M2].

OBJECTIVE: To summarize the clinical characteristics of patients with acute myeloid leukemia-type M2 (AML-M2) and analyze the factors affecting the prognosis. METHODS: One hundred eighty-eight AML-M2 patients were retrospectively analyzed for the following parameters including peripheral blood, immune phenotypes, fusion genes and cytogenetics to explore their significance for the overall survival (OS) and progression-free survival (PFS). The prognostic factors were also analyzed. RESULTS: Among 188 patients with AML-M2, the chromosomal abnormality with t (8;21), normal chromosome and other abnormalities accounted for 37% (70/188), 41% (77/188) and 22% (41/188), respectively. For the immunopheno typing of M2 patients, the hematopoietic progenitor cell differentiation antigen CD117 (96.1%) were mainly expressed, CD34 (81.6%) and HLA-DR (55.9%), and myeloid-associated antigen of CD13 (90.5%) and CD33 (89.4%) were also highly expressed. There were lymphoid-associated antigens expressed in some patients, among which the positive expression rate of CD19 was highest (29.6%), and the next was CD7 (28.5%). The most common accompanied mutations was FLT3 mutation (30.2%). The univariate analysis showed that the patients at age＜50 years old, without extramedullary infiltration, with positive expression of CD19, NPM-1 (-), CEBPA double mutation(+), and HSCT were significant superior in OS and PFS (P＜0.05); the multivariate analysis showed that the patient at age＜50 years old, without extramedullary infiltration, with positive expression of CD19 and CEBPA double mutation (+) were significant superior in OS and PFS (P＜0.05). The analysis indicated that the Karytypes affected only OS (P＜0.05), while the NPM-1 gene mutation positive affected only PFS (P＜0.05). The univarate analysis of factors affecting the survival in 70 AML-M2 patients with t (8;21) abnormatity showed that the C-KIT gene mutation was a poor factor for OS and PFS. CONCLUSION: The clinical characteristics are different between M2 patients with different karyotype, and prognostic analysis shows that the karytypes have an impact on overall survival; age, extramedullary infiltration, CD19 expression and CEBPA double mutation are also the main factors impacting the prognosis of patients.

[Differentiation of K562 Cells Induced by Pulsatilla Saponin A into Erythroid Lineage].

OBJECTIVE: To explore the differentiation-inducing potentiality of Pulsatilla saponin A on K562 cells. METHODS: Pulsatilla saponin A of different concentrations was used to treat K562 cells; the benzidine staining and the hemoglobinometry were applied to measure the change of hemoglobin content; the flow cytometry (FCM) was used to detect the expression of CD71 and GPA on K562 cells. RESULTS: K562 cells treated with 4 µg/ml pulsatilla saponin A differentiated into the erythroid lineage. With the treatment of pulsatilla saponin A, the hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the K562 cell surface were up-regulated. CONCLUSION: Pulsatilla saponin A can induce K562 cells to differentiate into erythroid lineage.

[Autophagy Activity of CD34+ Cells in MDS Patients and Its Clinical Significance].

OBJECTIVE: To explore the autophagy activity of CD34+ cells in bone marrow of MDS patients and its clinical significance. METHODS: 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). RESULTS: 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 (P<0.05), and more interestingly, the autophagy activity in MDS negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r=-0.877) . CONCLUSION: 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.

[Alteration of Microparticle Levels in Early Complications During Hematopoietic Stem Cell Transplantation].

OBJECTIVE: 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. METHODS: 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. RESULTS: (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 (P < 0.05). (3)The levels of TF(+) MP and EMP in thrombotic group at different times were significant differences from those in other groups (P < 0.05), and the levels of TF(+) MP and EMP were no significant difference from those in non-complication group. CONCLUSION: 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.

[Clinical and Laboratorial Characteristics of Primary Acute Myeloid leukemia with Philadelphia Chromosome and Inversion 16].

OBJECTIVE: 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). METHODS: A series of clinical tests, the cellular morphological, immunological, cytogenetic and molecular biological examinations of leukemia cells were performed. RESULTS: 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 CBFß allele was over that of the BCR-ABL fusion signals. The obvious adverse events did not occur in this patient within 3 years. CONCLUSION: 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.

[The variation and clinical significance of paroxysmal nocturnal hemoglobinuria clone in patients with aplastic anemia before and after immunosuppressive therapy].

OBJECTIVE: 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). METHODS: 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. RESULTS: 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 and > 24 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 values < 0.05). Clinical response rates were comparable in both ATG+CsA or CsA alone groups no matter PNH clone was positive or negative. CONCLUSIONS: 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.

[Clinical and laboratorial analysis for 15 adult cases of mixed phenotypic acute leukemia with Ph chromosome and/or positive BCR-ABL].

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.

[Changes of transcription factors Bcl-6, Foxp3 and RORγt in CD4(+) cells in bone marrow of immuno-related hematocytopenia].

OBJECTIVE: 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). METHODS: 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). RESULTS: 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), P < 0.05). The expression of Bcl-6 was higher (2.243 (0.854-4.544) vs 1.211 (0.131-2.816), P < 0.05). 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), P < 0.01) 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), P < 0.01). CONCLUSION: 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.

Growth and differentiation effects of Homer3 on a leukemia cell line.

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 As2O3 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.

Detection of autoantibodies against platelet glycoproteins in patients with immune thrombocytopenic purpura by flow cytometric immunobead array.

BACKGROUND: 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). METHODS: 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. RESULTS: 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 values<0.01). 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, p<0.01). CONCLUSION: FCIA assays with multiple antibodies against platelet GPs may be used to improve the diagnosis of ITP in hospitals.

[Detection of tissue factor-positive microparticles and its clinical significance in the haemostatic disorder].

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 (P < 0.05). The TF(+) MP level was higher in the patient with APL than that in the healthy adults, especially in course before therapy (P < 0.01), 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 (P < 0.05), 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.

[Analysis of immunophenotype characteristics in 109 cases of B lineage chronic B lymphoid leukemia].

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.

[Study on the clinical characteristics of 32 patients with mixed phenotype acute leukemia].

OBJECTIVE: To investigate the clinical and biological characteristics and prognosis of mixed phenotype acute leukemia (MPAL). METHODS: 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). RESULTS: (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. CONCLUSIONS: 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.

[Detecting minimal residual disease status in allogeneic hematopoietic stem cell transplantation of patients with high-risk acute leukemia].

OBJECTIVE: 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). METHODS: 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). RESULTS: 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%, P < 0.05) and 3-year RFS (85.71% vs 44.72% vs 68.48%, P < 0.05). 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 (P < 0.05). CONCLUSION: 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.