Genetic analysis and immunophenotyping in the diagnosis of hematological disease

The developments in the fields of genetics and immunology and the application of these informations have significant consequences for the diagnosis of hematological diseases. The present article gives an introduction into the principles of several modern diagnostic techniques, which are applied in the diagnosis of hematological diseases. In addition, it summarizes the application of these techniques in the diagnosis of several acquired and inherited diseases. The most important method of immunophenotyping is FACS (fluorescence activated cell sorting) analysis, which is based on the automated recognition of fluorescently labelled monoclonal antibodies bound to specific antigens on the surface or in the cytoplasm of different cell populations of the immune system. Techniques from molecular biology and from cytogenetics are also relevant for the diagnosis of hematological diseases: they allow the identification of changes of the genetic material on the level of DNA (molecular biology) and chromosomes (cytogenetics). Molecular biological and cytogenetic methods coalesce in the field of molecular cytogenetics, which renders possible the identification of chromosome mutations, which are invisible by the classical cytogenetical approach, and difficult to detect by routine molecular biological analysis. Most hematological malignancies are associated with genomic changes, which can be identified by cytogenetic and/or molecular biological methods. These genetic changes usually correspond with a specific pattern of surface antigens of the tumour cells, which can be identified by FACS. The different mutations in different genes causing a large number of inherited hematological diseases can often be found by molecular analysis, too. For hematological neoplasias, the exact definition of the causative mutations is increasingly important for therapeutic decisions and follow-up analysis of minimal residual disease. For inherited diseases, the identification of mutations is often the basis for a correct genetic counselling of the family.     

急性白血病免疫表型的预后意义

免疫表型对白血病的预后判断有重要意义。目前临床宜选用一些与细胞分化阶段有明确关系的、代表某些形态学特征的抗原进行免疫分型,可补充FAB分型的不足,还可确定形态学不能或很难区分的白血病类型。通过免疫表型检测和计分可明确双表型白血病和未分化白血病的诊断。某些特异的抗原与特异的细胞遗传学以及分子水平异常有关,决定了白血病的恶性行为及对治疗的反应。用多参数流式细胞术可同时检测2个或更多的抗原,数种抗原组合可用来检测微小残留病,提高检测的准确性,在患者初诊时即对其各白血病细胞亚群进行表型分析,为以后的微小残留病监测提供依据,才具有预测复发的价值。     

单体7或7号染色体长臂缺失的意义

本文先介绍带有失去全部染色体7(-7)或失去7号染色体长臂片段(7q-)的痰病,包括MDS和AML,原发性-7(de novo—7),蛙发性髓系疾病伴有-7者,体质性疾病伴有-7者。本文又论速-7细胞的生物学特性,包括-7发生于哪个阶段的祖细胞,带有-7的祖细胞生长的特点、-7细胞遗传学的异常和缺失的图谱,儿童白血痛前期RAS旁路的突变和7q-断点的分析等。总结这些材料,提示先检查患者的染色体,查明缺失的片段和确切的断点,再分析缺失片段厦断点附近的基因井进行克隆,由此可能分离出AML和MDS位于7号染色体的押癌基因,可能为白血病的发病和治疗研究提供新的策略。     

BCR-ABL融合蛋白多样性及其与白血病表型的关系

本文报告了BCR-ABL融合蛋白及其与白血病表型的关系。首先报告BCR-ABL融合基因的结构及其转录本,其中包括这些融合基因可分为M-bcr,m-bcr和u-bcr三种类型及其见于那些白血病;其次报告了BCR断点位置的变化,ABL的特殊断点,BCR-ABL信息RNA的拼接,BCR-ABL融合蛋白的结构与白血病表型的关系;此外,还介绍了BCR/ABL细胞有染色体的其它异常和Ph染色体的变种等;最后论述了BCR/ABL融合蛋白起源于何种细胞系,何种成熟阶段。少数文献报告此蛋白起始于多能干细胞。     

Current methods of molecular cytogenetics in pre- and postnatal diagnosis of chromosome aberrations

Progress in prevention of chromosome aberrations is due to utilization of molecular cytogenetic diagnostic methods. The purpose of this trend of clinical cytogenetics is development and utilization of new highly effective methods for analysis of chromosome aberrations. Molecular cytogenetic methods (fluorescent in situ hybridization-FISH) are used for pre- and postnatal identification of chromosome aberrations in mentally retarded children and congenital diseases. These studies are carried out after classical cytogenetic analysis, if it proves to be of no avail. FISH diagnosis pre- and postnatally detects autosomal trisomy, gonosome aneuploidy (including mosaic forms), marker chromosomes, structural chromosome aberrations, including fragile X chromosome syndrome. Rapid (15-30 min) FISH with an original collection of centromere, telomere, and site-specific DNA probes (plasmid, cosmid, PAC and YAC clones) is recommended for molecular cytogenetic diagnosis. FISH diagnosis is an effective complex of methods for pre- and postnatal identification of chromosome aberrations and a necessary supplement to classical cytogenetic diagnosis. Molecular studies of chromosome aberrations are significant for theoretical and applied studies, for they help detect patients with specific chromosome syndromes from a vast group of children with undifferentiated mental retardation and congenital diseases.     

Acute myeloid leukemia: a classification and treatment update

Approximately 11,000 Americans will be diagnosed with acute myeloid leukemia (AML) in 2003, and about 75% ultimately will die from the disease. Despite significant advances in understanding biologic, molecular, and cytogenetic aspects of this malignancy, several other areas remain poorly understood. During the 1990s, significant advances in the characterization of this condition have shown that AML affects elderly patients more frequently. Treatment of patients in this age group poses a greater challenge partly because of increased tumor resistance and the presence of multiple medical comorbidities that may contraindicate therapy. New therapeutic approaches are promising and have renewed enthusiasm and optimism among patients and healthcare providers. Future treatment strategies for patients with AML most likely will include combinations of biologic agents with defined molecular targets (e.g., monoclonal antibodies, retinoids, hypomethylating agents, tyrosine kinase inhibitors).     

Review: progress of cytogenetics in hematopoietic malignancies]

In 1960, Nowell and Hungerford found, for the first time, a minute chromosome at the metaphase in chronic myelocytic leukemia (CML) cells, which was called Philadelphia chromosome (9; 22 translocation) later. Ph1 chromosome was considered to be specific for the disease and was frequently used as an important marker for the definite diagnosis. In 1970s banding techniques revealed some other specific chromosome abnormalities, like 8; 14, 8; 21, and 15; 17 translocations, for acute leukemias. In 1980s, molecular-biology techniques were applied in the fields of leukemia research. As a result, many break point cluster regions were discovered in relation to the immunoglobulin chain genes and T cell receptor genes (Table 2). In this review, the specificity of chromosome abnormalities as well as genetic changes in types of leukemia is discussed.     

BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor alpha chimeric protein (PMLRARalpha) to block neutrophil differentiation and initiate acute leukemia

The promyelocytic leukemia retinoic acid receptor alpha (PMLRARalpha) chimeric protein is associated with acute promyelocytic leukemia (APL). PMLRARalpha transgenic mice develop leukemia only after several months, suggesting that PMLRARalpha does not by itself confer a fully malignant phenotype. Suppression of apoptosis can have a central role in tumorigenesis; therefore, we assessed whether BCL-2 influenced the ability of PMLRARalpha to initiate leukemia. Evaluation of preleukemic animals showed that whereas PMLRARalpha alone modestly altered neutrophil maturation, the combination of PMLRARalpha and BCL-2 caused a marked accumulation of immature myeloid cells in bone marrow. Leukemias developed more rapidly in mice coexpressing PMLRARalpha and BCL-2 than in mice expressing PMLRARalpha alone, and all mice expressing both transgenes succumbed to leukemia by 7 mo. Although both preleukemic, doubly transgenic mice and leukemic animals had abundant promyelocytes in the bone marrow, only leukemic mice exhibited thrombocytopenia and dissemination of immature cells. Recurrent gain of chromosomes 7, 8, 10, and 15 and recurrent loss of chromosome 2 were identified in the leukemias. These chromosomal changes may be responsible for the suppression of normal hematopoiesis and dissemination characteristic of the acute leukemias. Our results indicate that genetic changes that inhibit apoptosis can cooperate with PMLRARalpha to initiate APL.     

Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse

Genomic studies in human acute lymphoblastic leukemia (ALL) have revealed clonal heterogeneity at diagnosis and clonal evolution at relapse. In this study, we used genome-wide profiling to compare human T cell ALL samples at the time of diagnosis and after engraftment (xenograft) into immunodeficient recipient mice. Compared with paired diagnosis samples, the xenograft leukemia often contained additional genomic lesions in established human oncogenes and/or tumor suppressor genes. Mimicking such genomic lesions by short hairpin RNA-mediated knockdown in diagnosis samples conferred a selective advantage in competitive engraftment experiments, demonstrating that additional lesions can be drivers of increased leukemia-initiating activity. In addition, the xenograft leukemias appeared to arise from minor subclones existing in the patient at diagnosis. Comparison of paired diagnosis and relapse samples showed that, with regard to genetic lesions, xenograft leukemias more frequently more closely resembled relapse samples than bulk diagnosis samples. Moreover, a cell cycle- and mitosis-associated gene expression signature was present in xenograft and relapse samples, and xenograft leukemia exhibited diminished sensitivity to drugs. Thus, the establishment of human leukemia in immunodeficient mice selects and expands a more aggressive malignancy, recapitulating the process of relapse in patients. These findings may contribute to the design of novel strategies to prevent or treat relapse.     

急性混合细胞白血病伴t(12;22)一例报告--附文献复习

目的报道1例伴有t（12;22）（p13;q12）的急性混合细胞白血病.方法骨髓细胞经24h短期培养后按常规方法制备染色体,采用R显带技术进行细胞遗传学分析.应用抗生物素蛋白生物素复合物（ABC）法和单克隆抗体检测白血病细胞的表面抗原;12号和22号全染色体涂染探针分别以绿色和红色2种荧光素标记后进行双色荧光原位杂交（FISH）涂染检测.结果患者的临床表现和实验室检查均符合急性混合细胞白血病.免疫表型分析髓系和淋系标记均呈阳性;染色体核型为46,XX,t（12;22）（p13;q12）[6]/46,XX,idem,der（2）[2]/46,XX[12].骨髓中期细胞经双色FISH证实12号染色体短臂和22号染色体长臂之间发生了易位.结论t（12;22）（p13;q11-13）是恶性血液病中少见的染色体异常.t（12;22）患者具有独特的临床、细胞遗传学和分子生物学特点.该染色体异常对急性白血病的预后判断价值仍需进一步观察.     

慢性髓系白血病急变期分子遗传学研究进展

9号和22号染色体相互易位产生Ph染色体及BCR-ABL融合基因,几乎在所有慢性髓系白血病（CML）出现,BCR-ABL编码的蛋白具有持续增高的酪氨酸激酶活性,使白血病细胞异常增殖。急变期是CML的晚期,在此期间常常出现其它附加染色体和分子的改变。大量研究表明,BCR-ABL基因与其他失调的基因共同作用并异常激活下游的信号传导通路,促进了疾病的进展。酪氨酸激酶抑制剂伊马替尼对大多数慢性期CML患者治疗效果显著。IRIS5年的临床试验显示：用伊马替尼治疗的98%患者达血液学完全缓解,92%患者达主要细胞遗传学缓解,87%患者达完全细胞遗传学缓解。然而,仍有少数慢性期和大多数进展期患者用伊马替尼治疗疗效欠佳。在耐药机制的研究中发现ABL激酶区点突变与临床耐药关系密切。第二代酪氨酸激酶抑制剂可改善伊马替尼耐药,本文就急性变的分子机制、伊马替尼耐药等做一综述。     

实时定量逆转录PCR在白血病标志物检测中的应用

目的 建立实时定量逆转录PCR（RQ-RT-PCR）检测各种急慢性白血病的特征性分子生物学标志物,评价其在疾病诊断和微小残留病（MRD）监测中的意义.方法 设计TaqMan探针和引物,建立RQ-RT-PCR法对各种融合基因转录本和ABL阳性模板进行扩增,并检测177份白血病标本的转录本含量,同时做细胞遗传学检查.结果 RQ-RT-PCR法最低可检测到10个拷贝/μl的阳性模板,但重复性较差,而（10^8～10^2）拷贝/μl的重复性良好,正常对照无扩增信号.与细胞遗传学相比,RQRT-PCR的敏感性和特异性更强,对白血病标志物的阳性检出率更高.对12例不同类型白血病患者的MRD随访监测表明：患者体内融合基因转录本的含量随病情进展逐渐升高,随病情好转逐渐下降,并且早于细胞遗传学预测疾病复发.结论 RQ-RT-PCR方法更敏感、可靠,与疾病的关系更密切,可用于白血病的诊断和MRD随访.     

397例急性白血病细胞遗传学与FAB分型相关性研究

本研究探讨急性白血病的细胞遗传学改变,了解染色体核型异常与急性白血病FAB分型的关系及其对预后因素的影响。397例急性白血病患者于治疗前抽取骨髓标本,采用短期细胞培养法制备染色体标本,应用G显带技术进行染色体核型分析。结果显示,397例急性白血病中78例无分裂相,319例可供染色体核型分析的患者中染色体核型异常175例(占54.9%)。在急性淋巴细胞白血病(ALL)、急性髓系白血病(AML)和急性混合细胞白血病(AMLL)3种类型的白血病患者中,染色体核型异常分别为33/120例(占27.5%)、129/252例(占51.2%)和13/25例(占52.0%)。超二倍体41例(占23.4%),亚二倍体22例(占12.5%),正常二倍体112例(占64.0%)。AML染色体核型异常中与FAB分型相关的特异性染色体重排74/129例(占57.4%)。结论:大约55%左右的急性白血病存在克隆性染色体异常,一些特异性染色体异常改变是急性白血病的细胞遗传学特征,与急性白血病的FAB分型有明显相关性,染色体检查和分子遗传学方法相结合,对于白血病的诊断、分型、治疗和预后都具有重要意义。     

New aspects of preleukemic disorders.

Preleukemic disorders are a controversial group of panmyelopathic disturbances that often precede the emergence of acute myeloblastic or myelomonocytic leukemia. In most instances, these preleukemic disorders are characterized by slowly developing myeloblastosis of the bone marrow. They include preleukemia, primary acquired panmyelopathy with myeloblastosis or smouldering acute leukemia, erythroleukemia, and subacute myelomonocytic leukemia. Sometimes, transitions between these various preleukemic disorders may be observed in a single individual. Abnormalities in cellular differentiation are expressed in cytochemical aberrations and in elaboration of colony forming units by marrow cells of patients with preleukemic disorders. Cytogenic and cellular kinetic abnormalities link preleukemic disorders closely to acute myeloblastic or myelomonocytic leukemia, although in many patients with preleukemic disorders, conversion to acute leukemia is not observed or perhaps not recognized. Understanding pathogenetic and pathophysiological aspects of preleukemic disorders may shed light on aspects of cellular proliferation and cellular differentiation in the acute leukemias.     

Leukemias of indeterminant lineage

Two biologically and clinically important forms of acute leukemia have been described. Evidence suggests that both undifferentiated acute leukemia and many types of hybrid leukemia arise from relatively fewer differentiated cells than do the more straightforward acute leukemias. Clinical correlations suggest that patients with these leukemias may have a poorer prognosis, although some findings may be associated with an improved prognosis. More data in which multiple techniques are applied to the same leukemic cells are clearly needed. Patients with certain types of hybrid leukemias may be suitable candidates for more aggressive forms of chemotherapy or, possibly, combinations of therapy directed at cells of both lineages.     

应用国产ES探针检测慢性髓系白血病的bcr/abl融合及der(9)中间缺失

本研究旨在验证国产LSI bcr/abl ES探针检测慢性髓系白血病(CML)bcr/abl融合基因及衍生9号染色体中间缺失的有效性。应用国产LSI bcr/abl ES探针对97例经骨髓细胞形态学及常规细胞遗传学显带分析确诊的CML患者进行FISH检测,对具有der(9)中间缺失的病例再进行ASS基因探针的FISH检测,并取同期129例染色体核型正常的除外血液肿瘤性疾病及骨髓增殖性疾病患者作为阴性对照。结果显示:97例CML患者中91例染色体核型分析具有经典的t(9;22),6例为变异易位。经FISH检测所有患者均具有bcr/abl融合信号,其中16例具有der(9)中间缺失,占16.5%,在16例der(9)中间缺失的病例中13例具有ASS基因的缺失。129例阴性对照患者均未检测出bcr/abl融合。结论:国产LSI bcr/abl ES探针能有效识别bcr/abl融合及der(9)中间缺失,无假阴性及假阳性结果,ES-FISH检测结果与G显带核型具有很好的一致性。     

Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias

Free radicals are highly reactive species that have been implicated in the pathogenesis of many diseases. Reactive oxygen species can initiate lipid peroxidation and DNA damage leading to mutagenesis, carcinogenesis and cell death, if the antioxidant system is impaired. This study was undertaken to examine the prevalence of oxidative stress and the role of antioxidant defence in untreated leukemia patients. The generation of superoxide anion and hydrogen peroxide by leukocytes, plasma malondialdehyde levels, red cell copper zinc superoxide dismutase (Cu-Zn SOD) and glutathione peroxidase (GSH-PX) activities were determined in 30 patients with different types of leukemias prior to therapy. The superoxide anion generation by polymorphonuclear leukocytes was found to be significantly increased in leukemia patients especially those with acute lymphocytic and nonlymphocytic leukemias, while the hydrogen peroxide levels were comparable to the control values. Plasma lipid peroxidation products in untreated leukemia patients were in the normal range. Red cell Cu-Zn SOD and GSH-PX activities were significantly increased and showed no correlation with the hemoglobin content. Although superoxide generation was high, lipid peroxide levels were normal in these patients. This might be due to the increased activities of the antioxidant enzymes (SOD, GSH-PX) which counteract lipid peroxidation. Increased free radical generation, especially superoxide anion in leukemia patients and increased antioxidant defence enzymes, which is an adaptive protective response, are indicative of mild oxidative stress. There were no significant differences for the parameters cited above between different types of leukemias, suggesting that the changes are not specific to the type of leukemia.     

Cytogenetic and molecular detection of residual leukemic cells after allogeneic bone marrow transplantation in chronic granulocytic leukemia.

Allogeneic bone marrow transplantation (BMT) is a therapeutic modality with a curative potential for chronic granulocytic leukemia. Approximately 20% of patients have a hematologic relapse after BMT. The frequency of cytogenetic or molecular relapse (or both), despite hematologic remission, is reportedly higher. We performed allogeneic BMT in 32 patients with chronic granulocytic leukemia by using unmanipulated donor marrow and a conditioning regimen that consisted of cyclophosphamide and total-body irradiation. Of these 32 patients, 23 had cytogenetic studies after BMT. Seven of these patients had cytogenetically detectable Philadelphia chromosomes some time after BMT, during hematologic remission. The Philadelphia chromosome was detected transiently in two patients, and the fraction of abnormal metaphases exceeded 25% in three patients. None of the patients with negative results of cytogenetic studies or with the presence of the Philadelphia chromosome in less than 25% of analyzed metaphases had a clinical relapse, whereas two of the three patients with more than 25% abnormal metaphases had clinical relapses. Our results suggest that the detection of more than 25% abnormal metaphases during cytogenetic studies for chronic granulocytic leukemia after BMT may imply an incipient clinical relapse. We review the current literature that discusses isolated cytogenetic or molecular relapses of chronic granulocytic leukemia after BMT.     

Chromosome abnormalities and prognosis in non-Hodgkin's lymphoma]

Clonal chromosome abnormalities are found in most patients with non-Hodgkin's lymphoma. The role of the chromosome abnormalities in predicting the prognosis of lymphoma patients has not been fully clarified, because of different histological classifications being used in different areas and the complexity of the chromosome abnormalities often found in lymphoma. Recent studies have shown distinct correlation of rather rare abnormalities with specific histologic and immunologic phenotypes and prognoses. Many chromosome abnormalities seem to specifically correlate with these parameters of non-Hodgkin's lymphomas, as seen in leukemias. The chromosome data also seem to support the histological observations that some lymphomas may show uneven geographical distributions.     

Cell surface antigens of murine leukemias induced by radiation leukemia virus. Recognition of individually distinct cell surface antigens by cytotoxic T cells on leukemias expressing crossreactive transplantation antigens

The specificity of transplantation immunity and T cell cytotoxicity against leukemias induced by RadLV was examined. Subcutaneous inoculation of two RadLV leukemias induced in BALB/c mice, BALBRVB and BALBRVD, resulted in initial tumor growth in CB6F1 mice, followed by complete tumor regression. Mice that had rejected leukemias BALBRVB or BALBRVD were subsequently challenged with various tumors of BALB/c origin. The growth of all five RadLV leukemias tested, and of one radiation-induced leukemia, was significantly inhibited. Another radiation-induced leukemia, a methylcholanthrene-induced sarcoma, and a leukemia induced by the Moloney leukemia virus, were not inhibited. The results indicate that RadLV leukemias share cell surface antigens that induce transplantation immunity in vivo. Cytotoxic lymphocytes were generated by coculturing spleen cells from mice that had rejected leukemia BALBRVB or BALBRVD with the corresponding leukemia cells. Direct tests and inhibition tests showed that such cytotoxic cells recognized individually specific antigens on leukemias BALBRVB and BALBRVD, distinct from the shared antigens detected in transplantation experiments. The effector cells in cytotoxicity assays were Thy-1+, Lyt-1+,-, Lyt-2+, and Lyt-3+ T cells.