急性非淋巴细胞白血病的细胞和分子遗传学研究进展

随着细胞与分子遗传学技术在急性白血病(AL)的研究进展中的运用,AL的诊断分型已由1976年提出的以形态学为主的FAB分型发展到2001年的MICM分型,突出体现了细胞和分子遗传学的改变在AL诊断分型的重要地位。除此之外,细胞和分子遗传学改变还对急性白血病的危险度分层、预后判断、治疗指导及新药研发起着重要的指导作用。本文将论述染色体异常和融合基因的表达对急性非淋巴细胞性白血病(ANLL)的诊断分型、预后评估和治疗的指导作用,并对染色体核型正常的ANLL几种常见的基因突变对疾病预后评估做个总结。近年来,对ANLL各型白血病分子细胞遗传学机制的进一步阐释有助于开发新的白血病治疗策略。     

Morphology and classification of acute myeloid leukemias

The morphology and classification of AML are discussed. In addition to routine morphology and cytochemistry, however, it is now necessary to use newer modalities of immunocytochemistry or flow cytometry to confirm a diagnosis. These latter are essential for the diagnosis of the newer described entities of AML with minimal differentiation (FAB-M0) and acute megakaryoblastic leukemia (FAB-M7). Cytogenetics and fluorescent-in-situ-hybridization techniques are also very important for diagnosis as in FAB-M3 (promyelocytic leukemia) but also for detecting those myeloid leukemias that are associated with a favorable or unfavorable response.     

Molecular diagnosis in pediatric acute leukemias

This article summarizes the tremendous progress currently achieved in understanding the molecular basis of the pediatric acute leukemias. The article is organized from the perspective of the most frequently encountered pediatric acute leukemia genetic abnormalities in a molecular diagnostics laboratory setting. For each specific entity, the basic molecular biology, putative mechanisms of leukemogenesis, detection methods, and clinical significance are reviewed. Emphasis is placed on discussing the fusion genes generated from common nonrandom chromosomal translocations in B-lineage acute lymphoblastic leukemia (ALL), although brief summaries of T-lineage and myeloid leukemia, as well as the use of the antigen receptor gene rearrangement for residual disease monitoring in acute lympocytic leukemia are also presented. Finally, an overview of emerging technologies of potential importance in the laboratory diagnosis and evaluation of the pediatric acute leukemias is provided.     

Chronic myeloid leukemia: current application of cytogenetics and molecular testing for diagnosis and treatment

Chronic myeloid leukemia provides an illustrative disease model for both molecular pathogenesis of cancer and rational drug therapy. Chronic myeloid leukemia is a clonal stem cell disease caused by an acquired somatic mutation that fuses, through chromosomal translocation, the abl and bcr genes on chromosomes 9 and 22, respectively. The bcr/abl gene product is an oncogenic protein that localizes to the cytoskeleton and displays an up-regulated tyrosine kinase activity that leads to the recruitment of downstream effectors of cell proliferation and cell survival and consequently cell transformation. Such molecular information on pathogenesis has facilitated accurate diagnosis, the development of pathogenesis-targeted drug therapy, and most recently the application of molecular techniques for monitoring minimal residual disease after successful therapy. These issues are discussed within the context of clinical practice.     

Chronic Lymphocytic Leukemia: Diagnosis and Treatment

The complexity of the treatment of patients with chronic lymphocytic leukemia has increased substantially over the past several years as a consequence of the advent of novel biological agents such as ibrutinib, idelalisib, and venetoclax, as well as increasingly potent anti-CD20 monoclonal antibodies. In addition, the identification of molecular predictive markers and the introduction of more sensitive and sophisticated techniques to assess minimal residual disease have allowed optimization of the use of chemoimmunotherapy and targeted therapies and may become standard of care in the future. This review summarizes the diagnosis, prognostication, and treatment of patients with chronic lymphocytic leukemia with emphasis on new prognostic and predictive factors and novel treatment strategies.     

从临床医生的角度看结核病诊断技术

结核病是全球最主要的呼吸道传染病之一。本文从一个临床医生角度对结核感染以及结核病的诊断技术进行了回顾,并分析了其优缺点。在结核感染诊断中,结核菌素皮肤试验(PPD)使用悠久,而γ-干扰素释放试验(IGRAs)较PPD更有优势。在结核病诊断技术中,主要讨论了细菌学、血清学、分子生物学以及病理学诊断技术。其中细菌学是最主要的诊断手段,主要用于传染源确定、敏感性判定、疗效评估等方面;血清学技术特异性、敏感性存在不足,不建议使用;分子生物学在结核病诊断中发展迅速,代表了未来一个方向;病理学传统与分子生物学技术的结合赋予其新活力。     

Myelodysplastic syndromes

The myelodysplastic syndromes are a diverse group of clonal stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenias, and an increased propensity to evolve to acute myeloid leukemia. The molecular pathogenesis of these disorders is poorly understood, but recurring chromosomal abnormalities occur in approximately 50% of cases and are the focus of much investigation. The availability of newer molecular techniques has allowed the identification of additional genetic aberrations, including mutations and epigenetic changes of prognostic and potential therapeutic importance. This review focuses on the key role of cytogenetic analysis in myelodysplastic syndromes in the context of the diagnosis, prognosis, and pathogenesis of these disorders.     

Principles and potentialities of the standardization of morphocytochemical diagnosis of acute leukemias

Under the present conditions, the competitive capacity of a health care facility is provided by the high level and timeliness of diagnosis of disease. The diagnosis of the types of acute leukemia (AL) may be accomplished immunologically, by using a 33-marker panel and without consideration of the morphocytochemical parameters of blast cells. But such an approach complicates and prolongs the examination of patients with AL. Moreover, morphocytochemical data more exactly define the stages of blast cell differentiation than does the immunological phenotype. Their preparation methods are simple and cost-effective. Only M0, M6, and M7 forms of leukemia require compulsory blast cell phenotyping, particularly in the differential diagnosis of acute myeloid leukemia and acute lymphoblastic leukemia. Search for new markers of leukemia cells, including lesions at the chromosomal or molecular levels, is under way. Some of them are only of theoretical value while other markers have been already used by hematologists to diagnose leukemia. Standardization in this essence is the self-assessment of a facility and reference comparison, which are based on the principles of its orientation to the patient, the adjusted system for controlling the quality of health care that is up to the world standards rather than the compliance with the state-regulated standard. The present paper discusses the ways of establishing the uniform rates and requirements for the morphocytochemical diagnosis of acute leukemias.     

Molecular and flow cytometry characterization during the follow-up of three simultaneous lymphoproliferative disorders: hairy cell leukemia, monoclonal B-cell lymphocytosis, and CD4(++) /CD8(+/- dim) T-large granular lymphocytosis--a case report

The simultaneous diagnosis of hairy cell leukemia and monoclonal B-cell lymphocytosis with the characteristics of "indolent" chronic lymphocytic leukemia is rare but not unknown. However, an association with a third clonal lymphoproliferative disorder has not previously been described. We report the simultaneous presence of hairy cell leukemia, monoclonal B-cell lymphocytosis, and alpha beta CD4(++) /CD8(+) T-cell large granular lymphocytosis in a 63-year-old man. After the diagnosis, the three lymphoproliferative disorders (i.e., two of B-cell lineage and one of T-cell lineage) were characterized by analysis of multiple sequential bone marrow and peripheral blood samples using flow cytometry and molecular techniques. We discuss these findings in the context of chronic antigen stimulation, immunosuppression, and apoptotic pathway alterations, which might be implicated in the accumulation of these abnormal clones in the same patient. Because the phenotype of the three clones is compatible with fully differentiated B lymphocytes (consistent with a postgerminal origin) and T-CD4(++) cells, we favor the possibility of an antigen-driven mechanism and a dysregulation of homeostatic apoptosis in this patient.     

利用PCR衍生技术检测血液系统疾病

PCR技术给血液病的诊断带来了革命性的进展,已经成为临床急性和慢性白血病基因分型,骨髓增殖性疾病确诊,海洋性贫血、血友病等多种血液遗传病的诊断不可或缺的基本技术.在PCR基础上,已经发展了包括多重巢式PCR、荧光实时定量PCR(Q-PCR)、变性高压液相色谱分析(DHPLC)、毛细管电泳、分子杂交和直接测序等方法.多重巢式PCR可检测多种白血病基因,确定白血病的类型.Q-PCR有效地解决了PCR污染的问题,可以监测白血病微小残留病.毛细管电泳法更适于基因片段长度有变化的基因突变.DHPLC适用于大批量标本的突变筛查.PCR产物的直接测序可靠性强,已经用于常规诊断.     

B-ALL相关的细胞和分子遗传学异常与B-ALL预后关系的最新研究进展

近年来,以危险度分级为基础的规范化治疗应用于白血病的临床诊治使得ALL的缓解率显著提高,但缓解后复发仍为其根治的重要难题.临床上经常使用染色体核型分析研究ALL的遗传学特点.细胞和分子遗传学异常作为白血病的特异标志,用于判断预后,从而制定有效且最适的治疗方案,并常用于追踪微小残留病变,因此有望成为白血病监测与治疗的新靶点.本文对B-ALL相关细胞和分子遗传学异常的结构和生理功能作简要介绍,重点对其与B-AL预后关系的最新研究进展作一综述.     

Molecular diagnostics of malignant disorders

Expansion of our understanding of the molecular basis of cancer has enabled us to apply molecular techniques to categorize malignancies into more uniform, informative groups. In this review we describe the basic molecular techniques used for this purpose, including Southern blotting, polymerase chain reaction (PCR) and quantitative PCR, fluorescence in situ hybridization, DNA microarrays, and proteomics. The main applications of these techniques in the modern management of acute leukemia, chronic myeloid leukemia, lymphomas, and breast cancer are summarized.     

Minimal residual disease in acute promyelocytic leukemia

In the last decade our understanding of acute promyelocytic leukemia (APL) has advanced tremendously. The recognition of all-trans retinoic acid (ATRA) as a powerful therapeutic agent paralleled the cloning of the t(15;17) breakpoint. RtPCR for the PML-RARA hybrid mRNA has become the hallmark of molecular diagnosis and molecular monitoring in APL. Current techniques are useful in predicting complete remission and a possible cure in many patients who repeatedly test negative by PCR. Standardizing techniques and improving the sensitivity of the assay are important. Doing this in a way so that clinically relevant minimal residual disease can be distinguished from "indolent disease" remains among the future challenges in APL.     

用分子细胞遗传学技术检测白血病染色体易位

本文概述了荧光素原位杂交(FISH),染色体涂抹(CP)和比较基因组杂交(CGH)等FISH相关技术,并讨论了这些技术应用于多种染色体易位的白血病的检测,其中包括对急性早幼粒细胞白血病(APL),儿童B系急性淋巴细胞白血病(B-ALL)及慢性粒细胞白血病急性变(CML-BT)病人的染色体易位的检测。这些技术的应用为白血病发病的分子机制的阐明奠定了基础,同时为临床诊断和残余白血病细胞监测提供了直观、快速、敏感和特异的方法。     

Development of acute myeloid leukemia from donor cells after allogeneic peripheral blood stem cell transplantation in a female patient with acute monoblastic leukemia

Development of leukemia from donor cells is a rare complication of allogeneic blood stem cells (BSC). The paper describes a case of evolving acute myeloid leukemia of a graft in a patient with resistant acute monoblastic leukemia after related allogeneic peripheral BSC transplantation. The rarity of this complication, difficulties in providing evidence for the donor origin of a leukemic clone demonstrate a need for all-round careful dynamic assessment of the hematopoietic system after allogeneic transplantation, by applying the current cytogenetic (fluorescence in situ hybridization) and molecular (hypervariable genomic region amplification test using the polymerase chain reaction, hypervariable number of tandem repeats (VNTR), and short number of tandem repeats (STR)) techniques, which permits errors to be avoided in the assessment of a clinical situation and in the diagnosis of leukemia from donor cells. There is no developed policy for treatment of acute graft-versus-leukemia.     

Imaging of early modification in cardiomyopathy: the doxorubicin-induced model

Doxorubicin chemotherapy is effective and widely used to treat acute lymphoblastic leukemia. However, its effectiveness is hampered by a wide spectrum of dose-dependent cardiotoxicity including both morphological and functional changes, affecting primarily the myocardium. Non-invasive imaging techniques are used for the diagnosis and monitoring of these cardiotoxic effects. The purpose of this review is to summarize and compare the most common imaging techniques used in early detection and therapeutic monitoring of doxorubicin-induced cardiotoxicity and the suggested mechanisms of such side effects. Imaging techniques using echocardiography including conventional 2D and 3D echocardiography along with MRI sequences including Tagging, Cine, and quantitative MRI in detecting early myocardial damage are also reviewed. As there is a multitude of reported indices and imaging methods to assess particular functional alterations, we limit this review to the most relevant techniques based on their clinical application and their potential to early detection of doxorubicin-induced cardiotoxic effects.     

Aptamers evolved from cultured cancer cells reveal molecular differences of cancer cells in patient samples

BACKGROUND: Molecular-level differentiation of neoplastic cells is essential for accurate and early diagnosis, but effective molecular probes for molecular analysis and profiling of neoplastic cells are not yet available. We recently developed a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy to generate aptamers (designer DNA/RNA probes) as molecular probes to recognize neoplastic cells. METHODS: We tested 6 cell-SELEX-generated aptamers with equilibrium dissociation constants in the nanomolar to subnanomolar range: sgd5, selected from Toledo cells, a human diffuse large-cell lymphoma cell line (B-cell), and sgc8, sgc3, sgc4, sgd2, and sgd3 from CCRF-CEM cells, a human precursor T cell acute lymphoblastic leukemia (T-ALL) cell line. Aptamers were labeled with fluorescein isothiocyanate fluorophores and then used to recognize, by flow cytometric analysis, neoplastic cells in cultured hematopoietic cell lines and clinical samples. RESULTS: Aptamer sgd5 recognized only its target cells. Aptamers sgc3, sgd2, sgd3, sgc4, and sgc8, selected from a T-cell leukemia cell line, identified all of the cultured T-cell leukemia cell lines with relatively high fluorescence intensity. Aptamers sgc8, sgc3, and sgd3 showed good selectivity toward T-ALL cells and almost no binding to normal hematopoietic cells or lymphoma and myeloma cells. Selected aptamers also detected targets on the cell membranes of neoplastic cells in patient samples. CONCLUSIONS: Aptamers selected against cultured neoplastic cells can effectively be used as molecular probes for recognition of neoplastic cells in patient samples. Cell-based aptamer selection can be used to generate aptamer probes to obtain molecular signatures of neoplastic cells in patient samples.     

The use of cytochemical procedures in the diagnosis and management of acute and chronic myeloid leukemia.

The use of Wright-Giemsa-stained smears alone for the classification of acute leukemias often proves unsatisfactory. Some cases of M1, M5a, M7, and L2 are morphologically similar. In such cases, cytochemical stains can provide an inexpensive and available diagnostic tool. M1 is positive for SBB and MPO. M5a is usually NSE positive, whereas SBB and MPO are negative. M7 usually is ANA esterase, PAS, and AP reactive, and do not stain with SBB, MPO, and ANB esterases. The megakaryocytic lineage usually is confirmed by ultrastructural cytochemistry for PPO or immunocytochemistry for platelet glycoproteins and von Willebrand factor. PAS block positivity and AP dotlike reactivity are suggestive of lymphoid lineage. NSE stains are useful in differentiating M2 from M4. Morphologic and cytochemical techniques also can suggest the presence of certain chromosomal abnormalities such as t(8;21) and inv(16), which may have an influence on prognosis. Because not all cases of acute leukemia are easily subtyped by morphology and cytochemistry, immunophenotyping, karyotyping, and molecular analysis of DNA and RNA of leukemia cells also may be required to define cell lineage.     

颗粒性急性淋巴细胞白血病实验室检查与分析

目的通过报道1例形态学较特殊的颗粒性急性淋巴细胞白血病（granular acute lymphocyte leukemia,G—ALL）,并就有关文献进行复习,以增加对本病的认识和提高诊断水平。方法采用MICM标准确诊;采用骨髓涂片（瑞氏一吉姆萨染色法）、细胞化学染色进行形态学检测;采用R显带技术进行细胞遗传学分析;多色流式细胞仪分析免疫表型并进行分子生物学检测。结果该例患者确诊为正常核型的普通B细胞型ALL。结论ALL诊断时应慎重,形态上需与大颗粒淋巴细胞白血病及反应性大颗粒或颗粒增多淋巴细胞增多症相鉴别。     

嗜碱性粒细胞白血病的诊断与治疗进展

嗜碱性粒细胞增多常见于慢性髓细胞白血病(CML)加速期或急变期,其变化可作为CML的预后评价指标.嗜碱性粒细胞白血病(BL)是以嗜碱性粒细胞异常增多为主要临床表现的独立白血病类型.临床在排除其他血液系统疾病后,依据骨髓检查及外周血检查结果将嗜碱性粒细胞异常增多引起的BL分为急性BL和慢性BL.因BL发病率较低,目前尚无明确的诊断标准.随着检测技术和诊疗水平的不断提高,基于形态学、免疫学、细胞及分子遗传学(MICM)的诊断模式,可使越来越多单独发病的BL被识别和发现.笔者拟就目前国内外发现的BL的诊断和治疗进展进行综述.