Breakthroughs in the immunotherapy of acute leukemia have been achieved in recent years. The paper summarizes the clinical results, experience, and prospect in this area. One of the most significant advancements is the finding that anti-CD19 chimeric anti-gen receptor T cells (CAR-T) or CD3/CD19 bispecific antibody increases the rate of complete remission in patients with refractory re-lapse acute B lymphocytic leukemia. The disease-free survival of patients with low or intermediate risk was dramatically improved by combining chemotherapy and adoptive cytokine-induced killer or natural killer cells. Many immunotherapy methods, such as those fo-cusing on other targets of CAR-T, T-cell antigen receptor-modified T cell, and other targets of bispecific antibodies, are currently being examined. Combined methods can further increase cure rate and improve patients' quality of life, decreasing application of allogeneic hematopoietic cell transplantation which increases risks and reduces the quality of life.

Remarkable achievements have been made for lymphocyte infusion of lymphoma and leukemia, especially of lymphoproliferative disease within twenty-first century. The donor lymphocyte infusion or mobilized donor lymphocyte infusion (DLI/DSI) offer an opportunity of second remission for relapse patients post-transplantation. Cytokine-induced killer/DC cytokine-induced killer play an anti-tumor activity beyond non-MHC restricted. Cytotoxic lymphocyte infusion activated by synthetic tumor antigen produces targeted effects of anti-tumor. Transgenic CTL of anti-tumor TCR has brought the dawn in lymphoma and leukemia patients with defective lymphocytes. Maternal lymphocyte infusion play an anti-tumor/viral activity by avoiding the immune barrier of HLA mismatch.

Chimeric antigen receptor T cell (CAR-T) therapy has shown promising perspective in clinical trails of B cell hematologic malignancies.Meanwhile,this therapy still need to be further improved in the following aspects:design of CAR-T,cancer antigens selection,T cells origin,and clinical application strategy.CAR-T immunotherapy is one of hot topics in the 56th American Society of Hematology (ASH) annual meeting.Some breakthroughs have been reported in both basic research and clinical trails.

Objective To study the value of flow cytometry in identifying metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Methods Twenty-six cell lines representing ten epithelial neoplasms, one lymphoma cell line and one human T cell lymphoblast-like cell line were purchased from American Tissue Culture Collection. From July 2009 to June 2010, five nonhematopoietic neoplasms,fifteen hematopoietic neoplasms and fifteen control patients with complete remession after hematopoietic stem cell transplantation were collected in Beijing Daopei Hospital. Cryopreserved cell lines were thawed and cultured until they entered log phase. After permeabilization, cell lines were analyzed by staining with cytoplasmic CK-FITC antibody using four-color flow cytometer. The percent CK positivity was measured by comparing with negative control. Bone marrow samples were stained with membrane and cytoplasmic antibodies according to our routine methods. Based on lineage markers and blast markers as well as CK expression, the relevant hematopoietic diseases were diagnosed or excluded according to 2008 World Health Organization diagnosis standards. Results All epithelial neoplasm cell lines expressed CK, with average positive percentage 81.1%. All the lymphoid tumor cell lines didn't express CK. Two epithelial neoplasms were CK positive, 100. 0% in thyroid carcinoma and 98. 2% in lung carcinoma, respectively. Hematopoietic tumor and control samples didn't express CK. They expressed relevant hematopoietic markers, such as CD45 as well as lineage markers, or CD138 and cytoplasmic immunoglobulin light chain. Three nonepithelial nonhematopoietic neoplasms didn't express CK. CK positive or negative nonhematopoietic neoplasms didn't express hematopoietic markers such as CD45, HLA-ABC and HLA-DR DP DQ, as well as lineage specific markers. Besides, CK positive might be helpful to suggest epithelial origin. Conclusion Flow cytometry with hematopoietic markers and CK can effectively exclude hematopoietic tumor and identify metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow.

ObjectiveTo understand the characteristics of mutations in BCR-ABL1 kinase domain mutation,these chronic myeloid leukemia (CML) and Ph positive acute lymphoblastic leukemia (ALL)patients who got imatinib treatment had poor effect.MethodsTotally 177 CML patients and 33 Ph( + )ALL patients were selected at Beijing Dao-Pei Hospital from Sep.2007 to Dec.2010.All of them were Chinese patients.Totally 243 bone marrow or peripheral blood specimens were collected from the patients,who had early effect,then resistance emergenced,or for more than 3 months of poor efficacy.Extracted total RNA from the specimens' nuclear cells,reversed transcription to cDNA.Amplified the whole span of BCRABL1 fusion kinase gene by nest PCR (from 242 to 493 amino acid coding sequence),used the type AB3130XL gene sequencing instrument determinate the gene sequence of ABL1 kinase region and then used the Variant Reporter V1.0 software to analyze the results of gene mutations.ResultsThirty-two kinds of different mutations were detected of ABL1 gene mutations,accounting for 34.2％ (83/243 cases).Among them,the T315I was 12％ (10/83),mutation rate was the highest,followed by Y253H was 11％ (9/83),G250E was 7％ (6/83),E255K was 7％ (6/83),M351T was 6％ (5/83),E459K was 5％ (4/83) ;Q252H,D276G,F317L,E355G,F359V,H396R were all 4％ (3/83).Three cases of insertion mutations were found,including 2 cases of 357-358insk,1 case of V304RfsX17.Seven patients had found existence two or more point mutations.The multiple drug resistance mutations might exist in the same leukemia clone.The same individual was not only contain common resistance mutations,but also rare point mutations,insertion mutations.The mutations might be lead to loss of kinase activity.ConclusionsUnder the imatinib drugs pressure,the ABL1 gene mutation in leukemia cells appears randomly,and results in different resistant clones.Different resistant clones can coexist in the same patients in vivo; resistant clones not only contain point mutations,but also contain inserted deletion mutations.

Objective To deepen the understanding of chronic eosinophilic leukemia (CEL).Methods The course of diagnosis and treatment in a case of FIP1L1/PDGFRα fusion gene negative CEL was reported. Flow cytometry was used to analyze the immunophenotype of the cells in peripheral blood and pleural fluid. Karyotype was analyzed with G-banding. The expression of FIP1L1/PDGFRα fusion gene was detected by RT-PCR technique. Routine pathological examination of the tissues from bone marrow, lung and spleen were performed. Result A sixteen-year-old girl had severe anemia, fever, splenomegaly,thrombocytopenia and dominant hypereosinophilia lasting for 22 months. Trephine biopsy showed a hypercellular marrow with eosinophilic proliferation and moderate reticular fibrosis. Eosinophilic infiltration was found in lung and spleen and embolism was also found in spleen. She had a clonal chromosomal abnormality t(5;12)(q31;p13). The expression of FIP1L1/PDGFRα was negative. An abnormal clone of T cells expressing CD3-,CD4-,CD8- was found in peripheral blood and pleural fluid, in which the cional T cell accounted for 5.43% and 1.66% of the total lymphocytes respectively. The patient was refractory to treatment with hydroxyurea, prednisone and interferon alpha. She had poor response to a combination of therapy with low dose cytosine arabinoside, mitoxantrone, vincristine, cyclophosphamide, methotrexate and prednisone. She did not respond to imatinib and died of multiple organ failure. Conclusion The present case fulfilled the WHO diagnostic criteria of FIP1L1/PDGFRα(-) CEL which did not respond to routine treatment and imatinib. Allogenic stem cell transplantation should be considered as early as possible in this case. It is noteworthy that clonal CD3-,CD4-,CD8- T-cell abnormality is related to the pathogenesis of CEL.

Objective To investigate early Epstein-Barr virus (EBV) reactivation and the outcome of preemptive therapy after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods From January 2007 to January 2009, totally 277 patients after allo-HSCT were studied (haploidentical 116,unrelated 75, matched sibling 86). Conditioning regimens were mainly busulfan (BU) + cyclophosphamide ( CY)/fludarabine(Flu) or total body irradiation (TBI) + CY/Flu. Antihuman thymocyte globulin (ATG)was added in haploidentical and unrelated transplants. Plasma EBV DNA was monitored once to twice weekly in the first 3 months after allo-HSCT with real time quantitative polymerase chain reaction (RQ-PCR). EBV viremia was diagnosed when EBV DNA was more than 5 × 102 copies/ml but without symptoms. Acyclovir (10 mg/kg, intravenous drip, 8 h) was used for preemptive therapy and immnuo-suppressants were decreased if possible. Results Totally 33 patients ( 11.9% ) developed EBV viremia with a median time at day 44 (day 19 to day 84). The incidences of EBV viremia in the transplants from matched sibling,haploidentical, unrelated donors were 0, 15.5%, 20. 0%, respectively. There was no significant difference between haploidentical and unrelated transplants ( P = 0. 09 ), but much less EBV viremia was seen in matched sibling transplant ( P = 0. 001 ). Twenty of 33 patients ( 60. 6% ) had complete response to preemptive therapy. The median time to reach EBV DNA negative in plasma was 11 (4-56) d. The median duration of preemptive therapy was 21 (14-60) d. Both univariate and multivariate analysis indicated that haploidentical and unrelated transplants, acute graft versus host disease (GVHD) were the risk factors for EBV viremia. Two-year overall survival in the patients with EBV viremia was significantly lower than that without EBV viremia (54. 2% vs 72. 1%, P = 0. 006 ). Conclusions Our large clinical study has demonstrated that preemptive therapy with acyclovir that is guided by EBV viremia is effective in majority of the patients with high-risk for EBV reactivation after allo-HSCT, which may further decrease the risk for developing life-threatening EBV disease or post-transplantation lymphoproliferative disorder. Haploidentical and unrelated transplants, acute GVHD are the risk factors for EBV viremia which has negative impact on survival.

Objective To develop a multiplex sequence-specific PCR assay for simultaneous screening of 5 types of JAK2 mutations and investigate its clinical application value. Methods Multiplex sequence-specific PCR assay for simultaneous screening of JAK2 V617F, K539L (include 2 types of gene mutations), N542-E543del and E543-D544del mutations were developed. 115 patients with myeloproliferative diseases (MPD) including 61 polycythemia vera (PV) cases, 43 essential thrombocythemia (ET) cases and 11 primary myelofibrosis (MF) cases were analyzed. Results The assay can screen the 5 types of JAK2 mutations efficiently. The detection sensitivity is 1% for JAK2 V617F mutation and 0.1% for the other mutations. JAK2 V617F mutation and JAK2 exon12 mutation were detected in 56 and 3 of the 61 PV samples, respectively. 27 of the 43 ET samples and 6 of the 11 MF samples were JAK2 V617F positive, but no JAK2 exon12 mutation was found in both groups. The 3 cases carrying JAK2 exon12 mutation had the clinical feature of erythrocytosis and erythropoietin-independent erythroid colony formation but without apparent leukocytosis, thrombocytosis and splenectasis. Conclusion The assay can simultaneously screen 5 types of JAK2 mutations with high sensitivity and thus lead to an increased detection rate.

Objective To analyze the etiological factor and genetic feature of a familial hemophagocytic lymphohistiocytosis patient with PRF1 mutation (FHL2) with human herpesvirus 7 (HHV7)infection and its family constellation. Methods Clinical characteristics, laboratory examinations of a FHL2 case with HHV7 infection were reported. HHV1-HHV8 virus DNA was screened by PCR; NK cell function was analyzed by flow cytometry; PRF1 gene mutations were analyzed by PCR and direct sequencing, structure of mutant PRF1 proteins were analyzed using ExPasy and I-TASSER server and genetics pedigree were analyzed. Results The patient's HHV7 viral was detected positive with DNA copy number of 350/106 peripheral nucleated cells. Flow cytometry analysis showed decrease both in proportion of perforin positive NK cells and perforin protein expression. Genetic testing showed PRF1 biallelic heterozygote mutations (c. 503G ＞ A/p. S168N and c. 1177T ＞ C/p. C393R) and pedigree analysis showed they were inherited. The patient was then treated with antivirus therapy, dexamethasone and VP16 therapy, but only achieved partial response. The patient was then followed by human leukocyte antigen 10/10 allele identical nonconsanguinity allogeneic hematopoietic stem cell transplantations (allo-HSCT) and soon the successful implantation of donor hematopoietic cells and persistent recovery was achieved. The patient was now surviving without recurrence for 9 months after allo-HSCT. Conclusions FHL is prone to be misdiagnosed as lymphoma. Genetic analysis of related gene mutation and herpes simplex virus detection will help in early and accurate diagnosis. Allo-HSCT is a fundamental treatment of FHL.

BACKGROUNDTo investigate whether autologous lung cancer tissues derived gp96-peptide complex/dendritic cell vaccine could induce peptide specific cytotoxic T lymphocyte (CTL) response in vitro.

METHODSA patient's tumor-derived antigens including gp96-peptide complexes and tumor cell lysate were co cultured with DCs derived from the same patient's bone marrow blood mononuclear cells. The various antigen/DC vaccines were used to stimulate peripheral lymphocytes. Interferon-γ (IFN-γ) level of activated lymphocytes was detected by ELISA method and the Cr51 release test was performed to evaluate the gp96-peptide specific CTL response in three kinds of target cells including the primary cultured tumor cells, PG cells and K562 cells.

RESULTSIFN-γ could be observed from the supernate collected in all antigen groups after the cognate T lymphocytes were stimulated by various vaccines. The concentration of IFN-γ induced by gp96-peptide complexes/DC vaccine was higher than that of other groups. In addition, the killing effect of the activated T lymphocytes on patient's primary tumor cells was higher than that on PG and K562 cells.

CONCLUSIONSAutologous tumor-derived gp96-peptide complexes can induce a peptide complex specific CTL response, and the CTL response is significantly intensified after DCs are pulsed.