混合表型急性白血病32例临床研究

目的 分析混合表型急性白血病(MPAL)的临床、生物学特征和预后.方法 总结2003年1月至2009年6月收治的32例MPAL患者资料,分析其骨髓细胞形态学、细胞免疫表型、细胞遗传学和分子生物学特点.32例患者采用了兼顾髓系、淋系的联合化疗方案治疗,2例在缓解期进行了异基因造血干细胞移植(allo-HSCT).对患者进行疗效和生存分析.结果 ①MPAL在急性白血病中的发生率为2.6%,32例MPAL患者中髓系和B系(M/B)表型16例(50.0%),髓系和T系(M/T)表型14例(43.8%),髓系、B、T三系(M/B/T)表型和B/T系表型各1例(3.1%).②CD34和HLA-DR阳性率分别为87.5%和62.5%.③异常核型发生率为70.0%(30例中21例),涉及Ph、11q23和复杂核型在内的结构重排和(或)数目异常.④32例MPAL患者总完全缓解(CR)率75.0%,2年总生存(OS)率和无病生存(DFS)率分别为14.8%和14.2%.M/B和M/T表型CR率分别为75.0%和71.4%.M/B和M/T表型患者的OS和DFS率比较差异无统计学意义.2例allo-HSCT患者均未复发,生存期延长.结论 MPAL是一类异质性的急性白血病亚型,其不良预后可能与染色体异常发生率高、CD34高表达和易于髓外浸润等因素有关.兼顾髓系、淋系的联合化疗和包括HSCT的强烈治疗可能有助于提高疗效.

Abstract：

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-lympboid ( 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.③Abnormal 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 (DES) 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.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

急性微分化型髓细胞白血病转换为急性混合表型白血病一例诊断分析

目的 分析1例急性微分化型髓细胞白血病转换为不另作特定分类急性混合表型白血病的诊断过程,并探索其与其他急性微分化型髓细胞白血病和急性混合表型白血病病例的异同.方法 采用细胞涂片染色或化学染色方法对1例原发急性微分化型髓细胞白血病转换为不另作特定分类急性混合表型白血病病例进行细胞形态学分析;采用流式细胞术进行免疫表型分析;采用染色体G显带技术进行核型分析;应用RT-PCR技术进行融合基因的检测.并与2例急性微分化型髓细胞白血病和1例急性混合表型白血病进行实验室诊断结果比较,了解这一罕见的急性白血病发生转化的特征.结果 转换前急性微分化型髓细胞白血病在形态上表现为骨髓原始细胞占0.82,可见明显核仁,无Auer小体;免疫表型为造血相关抗原CD38和HLA-DR阳性,部分髓系抗原(CD13、CD56和CD11b)阳性,淋系抗原CD7阳性;其他髓系抗原(MPO、CD33和CD15)阴性,B系抗原(CD79a、CD19和CD22)阴性,T系抗原(胞内CD3、CD4和CD8)阴性.而转换后不另作特定分类急性混合表型白血病在形态上表现为骨髓原始细胞极度增生,占0.42,嗜酸粒细胞增多,嗜碱粒细胞可见;免疫表型为造血相关抗原CD38和HLA-DR阳性,髓系抗原(MPO和CD13)阳性,B系抗原(CD19和CD79a)阳性,T系抗原(胞内CD3)阳性,淋系抗原CD7阳性.对照组白血病具有典型的形态学和免疫表型特点,均未见异常染色体核型和融合基因.结论 该病例诊断复杂,临床少见,综合分析急性微分化型髓细胞白血病和不另作特定分类急性混合表型白血病的实验室特征对确诊十分重要,而免疫表型的变化是关键.

Abstract：

Objective To analyze the diagnostic process of a rare case of acute myeloid leukemia with minimal differentiation undergoing a lineage switch to mixed phenotype acute leukemia, NOS-rare types,and to investigate its difference from other acute myeloid leukemia and mixed phenotype acute leukemia. Methods Following tests were performed on the patient with switched mixed phenotype acute leukemia and three control leukemia patients ( including two acute myeloid leukemia with minimal differentiation and one mixed phenotype acute leukemia ). Cell morphology was analyzed by bone marrow smear and related cell chemical staining. Immunophenotyping of bone marrow was performed by flow cytometry ( FCM ). G-banding technique was used for karyotype analysis and RT-PCR was used for fusion gene detection. All the laboratory data of the switched patient were compared to that of three control patients in order to reveal the characteristics of such a rare phenotype switch in acute leukemia. Results Before switching, the morphology of acute myeloid leukemia with minimal differentiation demonstrated 0.82 blasts occurring in bone marrow, distinct nucleoli and absence of Auer rods. Blast cells expressed hematopoieticassociated antigens ( CD38, HLA-DR ), myeloid antigens ( CD13, CD56, CD11b ) and CD7. And these blasts were negative for MPO, CD33, CD15, CD79, CD19, CD22, cytoplasmic CD3, CD4 and CD8. After switching, 0. 42 blasts were found in bone marrow, showed eosinophilia and presence of basophile. Blast cells expressed hematopoietic-associated antigens ( CD38, HLA-DR ), myeloid antigens ( MPO, CD13 ),lymphoid antigens ( CD19, CD79a ,cytoplasmic CD3, and CD7 ). The control group showed typical morphology and immunophenotyping. No abnormal karyotype and fusion gene were detected. Conclusions It is a rare and complicated case that acute myeloid leukemia with minimal differentiation switched to mixed phenotype acute leukemia, NOS-rare types. The laboratory features, especially the change of immunophenotyping play an important role in the diagnosis.     

STUDY ON CELL ORIGIN OF MALIGNANT LYMPHOPROLIFERATIVE DISORDERS WITH POLYMERASE CHAIN REACTION

Genotype of IgH, TCRγ and TCR δ gene rearrangement in 42 cases of malignant lymphoproliferative disorders were studied by using polymerase chain reaction (PCR) technique. The results suggested that among the 23 cases, in which malignant cells expressed B-lineage cell surface markers, 20 showed IgH gene rearrangement and 11 had TCRγ gene rearrangement and / or TCRδ gene deletion. All the 11 cases expressed T-lineage cell differentiation antigens were found to have TCRγand TCRδ gene rearrangement or deletion and only one had IgH gene rearrangement. Double rearrangements of IgH and TCRγ genes were detected in all the 3 cases of T and B double-phenotype ALL. In the cases malignant cells did not express any lineage specific antigens while 4/5 had TCRγ gene rearrangement but all failed in IgH gene rearrangement. The relation of cellular differentiation origin and rearrangement of antigen receptor genes with clinical manifestations was discussed.     

T细胞受体γ基因和免疫球蛋白重链基因在急性淋巴细胞白血病细胞的重排及其在分型中的意义

用PCR扩增检测27例急性淋巴细胞白血病(acute lymphocytic leukemia,ALL)和1例T细胞淋巴瘤(T lymphocytic lymphoma,T-LL)的T细胞受体(Tcell receptor,TCR)γ基因和免疫球蛋白重链(immunoglobulin heavy chain,IgH)基因重排。TCRγ基因在9例T-ALL及2例未分型白血病病例中全部重排;在6例B-ALL中有1例重排;在10例C-ALL中有1例重排,而IgH基因在24例ALL病例中重排,其中7例为T-ALL,1例为未分型白血病。TCRγ和IgH基因在1例T-LL中均无重排,在T-ALL、B-ALL和C-ALL中均有交叉重排现象。我们探讨了这些结果在分型中的意义,同时还注意到,在28例中的10例病人存在IgH和TCRγ基因双重重排,如作为特异的双标记检测其微小残留白血病细胞,无疑会提高准确率。     

成人急性T淋巴细胞白血病T淋巴细胞受体β链基因重排的特点及其在微小残留病定量检测中的意义

目的 建立以TCR β基因重排为分子标志,基于ASO引物的RQ-PCR方法,检测成人T-ALL患者的TCR β基因重排,以利于动态监测患者的MRD.方法 20例成人T-ALL患者的初诊DNA标本经多重PCR检测方法设计38条引物,分成3管,分别扩增TCR β基因完全重排和不完全重排,克隆产物经电泳和双色基因扫描鉴定.对其中4例患者进行基因测序和序列比对分析,根据克隆特异性序列信息,利用软件设计4条ASO引物,作为RQ-PCR检测的上游引物,合成通用的Jβ下游引物和TaqMan探针,对患者的随访标本定量监测MRD.结果 20例患者中有17例可检测到克隆性TCR β基因重排,克隆检出率为85.0%(17/20).其中16例可检测到至少1个Vβ-Jβ完全重排(94.1%,16/17),7例患者有Dβ-Jβ的不完全重排(41.2%,7/17),完全Vβ-Jβ和不完全Dβ-Jβ重排之间的比例约为2∶1.双色基因扫描分析显示Jβ2家族的使用频率为73%,Jβ1家族的使用频率为27%,Jβ2家族的使用频率明显高于Jβ1家族.4例患者使用ASO引物RQ-PCR扩增TCR β重排靶基因的标准曲线的斜率为-3.60～-3.27,相关系数＞0.99,敏感性达4×10-5μg/μl,荧光背景信号不显示或较低.监测4例T-ALL患者在疾病治疗各随访时间点的TCR β重排靶基因水平,包括诱导完全缓解、巩固治疗等时间点,RQ-PCR显示其MRD水平随病情的缓解呈逐渐下降的趋势.结论 以克隆性TCR β基因重排为靶分子的特异性寡核苷酸引物RQ-PCR方法可对T淋巴细胞系统的恶性克隆进行准确定量,适用于成人T-ALL患者MRD的随访监测.     

CD45RO阳性的B细胞性淋巴瘤

目的 研究免疫表型异常淋巴瘤的起源。方法 从48例B细胞淋巴瘤中筛选出3例CD45RO、CD20和CD79a( )，CD3(-)的淋巴瘤。采用免疫组化、PCR和基因扫描技术对其免疫表型，免疫球蛋白重链基因重排(IgH)和T细胞受体基因重排(TCR)进行深入研究。结果 3例淋巴瘤均为结外淋巴瘤。1例为滤泡性淋巴瘤，2例为弥漫性大B细胞性淋巴瘤。PCR和基因扫描显示3例均有IgH克隆性扩增；PCR未显示TCR扩增；高敏感性基因扫描显示低峰值TCR克隆性扩增，提示可能为背景T细胞扩增。结论 CD45RO不是T细胞特异性抗原，CD45RO阳性细胞不能完全等同于T细胞，因此，在研究和临床病理诊断中应选用多种抗体配合使用。     

T cell receptor gamma and delta rearrangements in hematologic malignancies. Relationship to lymphoid differentiation.

We have studied recombinatorial events of the T cell receptor delta and gamma chain genes in hematopoietic malignancies and related these to normal stages of lymphoid differentiation. T cell receptor delta gene recombinatorial events were found in 91% of acute T cell lymphoblastic leukemia, 68% of non-T, non-B lymphoid precursor acute lymphoblastic leukemia (ALL) and 80% of mixed lineage acute leukemias. Mature B-lineage leukemias and acute nonlymphocytic leukemias retained the T-cell receptor delta gene in the germline configuration. The incidence of T cell receptor gamma and delta was particularly high in CD10+CD19+ non-T, non-B lymphoid precursor ALL. In lymphoid precursor ALL, T cell receptor delta was frequently rearranged while T cell receptor gamma was in the germline configuration. This suggests that TCR delta rearrangements may precede TCR gamma rearrangements in lymphoid ontogeny. In T-ALL, only concordant T cell receptor delta and gamma rearrangements were observed. Several distinct rearrangements were defined using a panel of restriction enzymes. Most of the rearrangements observed in T-ALL represented joining events of J delta 1 to upstream regions. In contrast, the majority of rearrangements in lymphoid precursor ALL most likely represented D-D or V-D rearrangements, which have been found to be early recombinatorial events of the TCR delta locus. We next analyzed TCR delta rearrangements in five CD3+TCR gamma/delta+ ALL and cell lines. One T-ALL, which demonstrated a different staining pattern with monoclonal antibodies against the products of the TCR gamma/delta genes than the PEER cell line, rearranges J delta 1 to a currently unidentified variable region.     

Rearrangement of variable region T cell receptor gamma genes in acute lymphoblastic leukemia. V gamma gene usage differs in mature and immature T cells.

Using probes recognizing variable regions (V gamma) and joining regions (J gamma) of the T cell receptor (TCR) gamma gene, we have analyzed the usage of V gamma genes in 24 patients with T cell acute lymphoblastic leukemia (ALL) and 36 patients with B-precursor ALL. In CD3- T-ALL derived from immature T cells, V gamma genes more proximal to J gamma were frequently rearranged; V gamma 8, V gamma 9, V gamma 10, and V gamma 11 were used in 19 of 24 rearrangements. In contrast, CD3+ T-ALL derived from a more mature stage of T cell ontogeny, showed a high frequency of rearrangements involving V gamma genes distal to J gamma; V gamma 2, V gamma 3, V gamma 4, and V gamma 5 were used in 17 of 25 rearrangements. In B-precursor ALL, no notable bias of V gamma gene usage was observed. This probably reflects the possibility that TCR genes may not rearrange according to a T cell hierarchy when under control of a B cell gene program. Furthermore, deletions of those V gamma genes located 3' to rearranged V gamma genes were observed in all patients analyzed. This supports the theory that loop deletion is a major mechanism for TCR-gamma gene rearrangement.     

异源双链鉴定法检测淋巴瘤IgH、TCR-β基因重排

目的建立更敏感、特异性更强的PCR方法检测淋巴瘤IgH和，TCR-β基因重排。方法采用标准PCR方法以及异源双链鉴定的改良方法检测55例淋巴瘤组织，全部病例均经组织学明确诊断，其中36例为B细胞性淋巴瘤，19例为T细胞性淋巴瘤。对照为5例反应性增生的淋巴结组织。结果用改良PCR方法检测淋巴瘤组IgH和TCR-β基因重排49／55例为阳性，较标准PCR方法电泳条带更清晰、更易于判定；5例反应性增生的淋巴结组织均为阴性。结论异源双链鉴定的改良方法提高了检测淋巴组织肿瘤性增生的敏感性和特异性，值得推广。