急性白血病MLL基因重排检测的临床意义

 目的　研究混合谱系白血病（MLL）基因重排在急性白血病（AL）中的发生率、产生融合基因的常见类型及其临床意义。方法　采用多重巢式RT-PCR法对109例AL患者进行免疫表型检测,分析MLL基因重排阳性患者的临床特征。结果　109例AL中7例发生MLL基因重排,发生率为6.4 %,其中AML 4例（1例为M2,1例为M4,2例为M5）, ALL 3例,均为B-ALL。多重巢式RT-PCR检测到的融合类型为MLL-AF4、MLL-AF6、MLL-AF9、MLL-AF10、MLL-ENL。MLL基因重排阳性患者外周血WBC高于MLL基因重排阴性患者,差异有统计学意义（P＝0.019）。结论　多重巢式RT-PCR是检测MLL基因重排快速有效的方法。伴MLL基因重排的AL患者WBC较高,预后差。     

急性髓系白血病混合谱系白血病基因重排的表达及临床特点

 目的 对急性髓系白血病（AML）进行混合谱系白血病（MLL）基因重排的实验室和临床特点研究。方法 以多重 RT-PCR方法对98例AML进行MLL基因重排检测,并结合流式细胞仪免疫表型检测,对其中10例MLL基因重排患者进行分析。结果 在98例AML中MLL基因重排者10例（10.2 %）,其中2例MLL／AF6,2例MLL／AF9,1例MLL／AF17、1例 MLI／ELL,2例MLL／AFl0, 2例dup11q23。MLL 基因重排患者中7例为M5,2例为M4,1例为M2。其中3例有CD10或CD22淋巴系抗原表达。10例MLL 基因重排患者中8例接受治疗,6例获得完全缓解,监测相应的MLL 基因重排发现,持续阴性者可长期存活,持续阳性者很快复发,阳性者经强化治疗后可转阴。结论 多重 RT-PCR是临床检测 MLL基因重排较为精确和灵敏的方法,MLL基因重排的监测在AML在预后判断和治疗方案的个体化选择方面有重要意义。     

多重巢式反转录-聚合酶链反应检测白血病相关融合基因及其临床意义

 目的　检测白血病相关29种染色体结构畸变形成的融合基因并对其在白血病MICM分型、危险度分组和微小残留病（MRD）检测中的临床价值进行分析。方法　采用多重巢式反转录-聚合酶链反应（RT-PCR）方法对141例白血病患者进行29种染色体结构畸变形成的融合基因进行分析,并与骨髓染色体检测结果进行对比分析。结果　141例白血病患者骨髓或外周血标本中, 66例（46.8 %）阳性,具有13种染色体结构畸变产生的融合基因, 包括PML／RARα、bcr-abl p210、bcr-abl p190、AML1／ETO、TEL／AML1、E2A／PBX1、MLL／AFX、MLL／AF6、MLL／AF9、dupMLL、MLL／ENL、CBFβ／MYH11、TLS／ERG等。在78例ALL和57例AML标本中,分别有27例（47.4 %）和33例（42.3 %）为阳性,染色体结构畸变产生的融合基因分别为7种和6种。结论　采用多重巢式RT-PCR 方法,可快速分析29种染色体结构畸变产生的融合基因,可同时筛选出29种急性白血病的常见染色体易位,帮助临床诊断、疾病危险度分组、预后判断和治疗后微量残留病的检测。     

多重巢式RT-PCR和染色体核型分析技术在急性髓系白血病中的联合应用

背景与目的:细胞遗传学分析在白血病的诊断和预后判断中有重要价值,但常规显带不仅耗时,且难以获得良好的分裂相;而聚合酶链反应具有灵敏、快速的特点.本研究探讨联合应用多重巢式RT-PCR和染色体核型分析,对急性髓系白血病(acute myeloid leukemia,AML)中融合基因的表达及其在各亚型的分布和克隆性染色体异常的检出情况.方法:采用多重巢式RT-PCR技术对60例AML病例进行检测,其中37例同时进行染色体R或G显带.结果:60例AML患者中检出融合基因28例(46.7%),包括AML1/ETO、PML/RARα、CBFβ/MYH11、MLL基因异常(包括MLL/AF6、MLL/AF9、MLL/AF10、MLL/MLL)、DEK/CAN、TEL/PDGFR、AML1/MDS1(EVI-1).同时进行染色体R或G显带的37例病例中有30例可供分析,其中14例(46.7%)检出染色体结构和数目异常;联合多重RT-PCR可使AML克隆性染色体异常检出率增至59.5%(22/37).结论:联合多重巢式RT-PCR和染色体核型分析技术可以提高克隆性染色体异常的检出率.     

急性淋巴细胞白血病常见融合基因检测的研究

 【摘要】　目的　探讨联合应用多重巢式反转录聚合酶链反应（RT-PCR）技术和染色体核型分析对急性淋巴细胞白血病（ALL）常见融合基因的表达和克隆性染色体异常的检出情况。方法　采用多重巢式RT-PCR技术对189例ALL患者进行检测,同时进行染色体R或G显带。结果　189例ALL患者中69例（36.5 %）检出10种融合基因（E2A-PBX1、TEL-AML1、bcr-abl、MLL-AF4、MLL-AF6、MLL-AF9、MLL-AF10、MLL-ELL、SIL-TAL1、TLS-ERG）,染色体R或G显带可供分析的152例中,86例（56.6 %）检出染色体结构和数目异常;二者联合可使ALL克隆性染色体异常检出率增至69.3%（131例）。90例成年ALL患者中33例（36.7 %）检测阳性,其中bcr-abl 22例,未见TEL-AML1,99例儿童ALL患者中阳性36例（36.4 %）,其中TEL-AML1 24例,bcr-abl 2例。成年人组与儿童组bcr-abl 、TEL-AML1的表达率差异有统计学意义（均P＜0.01）。MLL相关融合基因、E2A-PBX1、SIL-TAL1、TLS-ERG等表达率差异无统计学意义（均P＞0.05）。66例正常核型的ALL患者检测出有bcr-abl 、TEL-AML1融合基因的存在。结论 成年人和儿童ALL融合基因表达各有侧重,多重巢式RT-PCR可用于初诊时染色体畸变的筛选,可在核型正常的ALL患者中检出隐匿的染色体易位,提供与预后相关的重要信息。     

急性淋巴细胞白血病AML1基因重排与MLL基因丢失同时出现临床意义的探讨

目的：对急性淋巴细胞白血病（ALL）急性髓系白血病（AML1）基因重排与混合谱系白血病（MLL）基因丢失同时出现进行探讨。方法：在常规细胞遗传学（CC）分析基础上运用荧光原位杂交技术（FISH），采用多种位点特异性DNA探针（染色体全染、特殊位点和双色易位融合探针），对63例ALL患者（8例成人，55例儿童）进行分析。结果：63例ALL患者中有4例（6．3％）出现MLL基因重排，其中3例出现MLL基因丢失，1例出现MLL基因的移位即t（4；11），3例出现了MLL基因丢失的患者同时合并有AML1基因重排，2例为t（12；21）易位而形成的TEL／AML1融合基因，1例为AML1基因复制引起的环形21号染色体，即r（21）。55例儿童ALL中有15例（27．3％）出现AML1基因重排，即由t（12；21）易位而形成的TEL／AML1融合基因，TEL／AML1阳性患者免疫分型均为B细胞型，8例成人均无t（12；21）。结论：儿童ALL常合并有t（12；21），TEL／AML1融合基因的出现是预后良好的指标，而MLL基因重排的患者具有对常规化疗不敏感及预后不良的特点，两者同时出现说明白血病染色体重排、病理过程及影响预后因素的复杂性。     

常规细胞遗传学和荧光原位杂交方法检测14q32/IgH基因重排

  目的 比较常规细胞遗传学（CC）,间期荧光原位杂交（FISH）技术及连续R显带后FISH检测免疫球蛋白重链（IgH）基因重排的应用价值。方法 应用常规细胞遗传学及间期FISH分析血液系统肿瘤患者43例。结果 43例患者中14q32+／IgH+患者19例,14q32-／IgH+患者2例（4.7 %）,14q32+／IgH-患者3例（7.0 %）,14q32-／IgH-患者19例。部分病例CC与间期FISH方法检测14q32／IgH基因重排得到了不一致的结果。对5例患者进行连续R显带后FISH分析,对照核型和FISH图,能清楚地看到IgH基因易位涉及的染色体。结论 间期FISH可以提高14q32／IgH重排检出率,R显带后FISH可以帮助识别与14q32易位的伙伴染色体。     

儿童急性淋巴细胞白血病TEL-AML1融合基因检测

 目的　检测儿童急性淋巴细胞白血病（ALL）中TEL-AML1融合基因的阳性率,探讨TEL-AML1融合基因的检测方法及其临床应用价值。方法　在形态学、免疫分型、细胞遗传学基础上,采用巢式反转录-聚合酶链反应（RT-PCR）和荧光原位杂交技术（FISH）检测31例ALL患儿骨髓单核细胞中TEL-AML1融合基因。结果　巢式RT-PCR技术和FISH技术均可以显著提高TEL-AML1融合基因的检出率,应用上述两种方法,31例患儿中检测出7例TEL-AML1阳性,占儿童初发ALL的22.6 %（7／31）,在B系ALL中的阳性率为25.9 %（7／27）。结论　t(12;21)形成TEL-AML1融合基因是儿童ALL最常见的染色体易位,常规染色体核型分析极难发现,需用巢式RT-PCR或FISH等分子检测方法加以证实。     

急性髓系白血病17种基因异常的检测

目的:探讨应用多重巢式RT-PCR、荧光定量PCR、PCR-SSCP银染技术检测初诊急性髓系白血病(acute myeloid leukemia,AML)中17种基因异常表达及在各亚型的分布情况,为个体化治疗提供依据.方法:采用多重巢式RT-PCR检测融合基因,PCR检测FLT3-ITD,荧光定量PCR检测NPM1的突变类型(A、B、D、I和R)及C-kit/D816V,PCR-SSCP银染技术检测CEBPA.对140例初诊AML患者(APL除外)的骨髓进行17种基因异常分析,并与骨髓染色体检测结果进行对比.结果:在140例初诊AML患者中检出基因异常占69例(49.3％),其中包括FLT3-ITD、NPM1、C-kit/D816V、CEBPA、HOX11、CBFβ/MYH11、AML-ETO、MLL/AF6、MLL/AF10、dupMLL、EVI1.同时对140例初治AML患者采用G显带技术行染色体核型分析,128例获得可供分析的染色体核型,其中57例(44.5％)检出染色体结构和数目异常.PCR在急性髓系白血病基因检测中较染色体核型分析具有更高的检出率.结论:PCR协同染色体核型分析检测初诊AML患者的基因异常,能提高临床诊断率、指导疾病危险度分组,为判断预后及监测微小残留病提供更好的理论依据.     

二例伴有i(17q-)的急性早幼粒细胞白血病的临床和实验研究

  目的 探讨伴有i(17q-)的急性早幼粒细胞白血病（APL）的临床和实验室特征。方法 骨髓细胞经24 h培养后按常规方法制备染色体,用R显带技术进行核型分析,并用PML／RARα和HER-2探针进行荧光原位杂交（FISH）检测。用反转录-聚合酶链反应（RT-PCR）检测PML／RARα融合基因。结果 2例的临床和血液学改变符合AML-M3诊断。染色体核型分析揭示2例患者染色体均存在t(15;17)易位及i(17q-),并通过FISH检测加以证实。2例RT-PCR 均检测到了PML／RARα融合基因。结论 i(17q-)是APL中一种少见的染色体附加异常,其预后意义有待进一步讨论。     

Incidence of MLL rearrangement in acute myeloid leukemia, and a CALM-AF10 fusion in M4 type acute myeloblastic leukemia

To determine the incidence of the mixed lineage leukemia (MLL) gene rearrangements in acute myeloid leukemia (AML) without cytogenetically-detected 11q23 abnormalities, we screened 64 cases of AML at diagnosis for MLL rearrangement by FISH. Three cases (4.7%) had a MLL rearrangement detected; one was shown to have a cryptic t(11;22)(q23;q11) and another to have a t(9;11)(p21-22;q23) which had been missed by the conventional cytogenetic study. No 11q23 structural abnormality was visible in the third case. Twenty-six of the 64 cases were further studied by Southern blotting and DNA hybridization, and four of these cases (15%) were found to have MLL rearrangement: in three of these, FISH had not detected any abnormality. FISH was also used to confirm MLL involvement in eight cases of AML that had a cytogenetic abnormality at 11q23; in one of these, Southern blot did not show a rearrangement. The survival of patients with MLL abnormalities identified by cytogenetics, FISH and/or DNA analysis was significantly worse than that of patients without MLL abnormalities (event-free survival p = 0.016) although two patients with a t(9;11)(p21-22;q23) were long-term survivors, consistent with this particular translocation having a better prognosis. One further case with a cytogenetic abnormality close to 11q23 was studied; it was found to have a t(10;11)(p13;q21), and the breakpoints were shown by FISH to involve the Clathrin Assembly Lymphoid Myeloid (CALM) gene at 11q21 and the AF10 gene at 10p13. Our data confirm the value of combining cytogenetic, FISH and molecular analyses to define the incidence and precise nature of MLL and 11q23 abnormalities in AML.     

Comparison of cytogenetics, Southern blotting, and fluorescence in situ hybridization as methods for detecting MLL gene rearrangements in children with acute leukemia and with 11q23 abnormalities.

Bone marrow samples from 67 children with acute leukemia and with cytogenetic evidence of chromosome 11 band q23 (11q23) abnormalities were characterized by fluorescence in situ hybridization (FISH) and Southern blot analysis to determine whether FISH could reliably detect MLL gene rearrangements in this population. Among the 42 patients with acute lymphoblastic leukemia (ALL), MLL gene rearrangements were detected in cells from 23 patients (54.8%) by both FISH and Southern blot analysis. FISH identified allelic deletions of MLL gene in five of 12 patients (42%) with ALL and with deletion of 11q23. In 22 of 25 children (88%) with AML, FISH detected MLL gene rearrangements, whereas Southern blot analysis identified rearrangements in 24 of 25 patients (96%). For children with acute leukemia and with 11q23 abnormalities, we recommend that FISH be used for the rapid screening of MLL gene rearrangements and that Southern blot analysis be used for the definitive assessment of the MLL gene status.     

Identification of MLL partner genes in 27 patients with acute leukemia from a single cytogenetic laboratory

Chromosomal rearrangements involving the MLL gene have been associated with many different types of hematological malignancies. Fluorescent in situ hybridization with a panel of probes coupled with long distance inverse-PCR was used to identify chromosomal rearrangements involving the MLL gene. Between 1995 and 2010, 27 patients with an acute leukemia were found to have a fusion gene involving MLL. All seven ALL patients with B cell acute lymphoblastic leukemia were characterized by the MLL/AFF1 fusion gene resulting from a translocation (5 patients) or an insertion (2 patients). In the 19 AML patients with acute myeloblastic leukemia, 31.6% of all characterized MLL fusion genes were MLL/MLLT3, 21.1% MLL/ELL, 10.5% MLL/MLLT6 and 10.5% MLL/EPS15. Two patients had rare or undescribed fusion genes, MLL/KIAA0284 and MLL/FLNA. Seven patients (26%) had a complex chromosomal rearrangement (three-way translocations, insertions, deletions) involving the MLL gene. Splicing fusion genes were found in three patients, leading to a MLL/EPS15 fusion in two and a MLL/ELL fusion in a third patient. This study showed that fusion involving the MLL gene can be generated through various chromosomal rearrangements such as translocations, insertions and deletions, some being complex or cryptic. A systematic approach should be used in all cases of acute leukemia starting with FISH analyses using a commercially available MLL split signal probe. Then, the analysis has to be completed, if necessary, by further molecular cytogenetic and genomic PCR methods.     

Infant acute lymphoblastic leukemia - combined cytogenetic, immunophenotypical and molecular analysis of 77 cases.

We used karyotyping, fluorescence in situ hybridization (FISH), Southern blotting, and RT-PCR in order to analyze prospectively 77 infants (less than 1 year of age) with acute lymphoblastic leukemia for the occurrence of 11q23/MLL rearrangements and/or other cytogenetic abnormalities. Out of the 69 informative samples we found an 11q23/MLL rearrangement in 42 cases (61%). Regarding only pro-B ALL cases, the incidence of 11q23/MLL rearranged cases, however, reached more than 90% The infants were treated within the therapy studies ALL-BFM90, ALL-BFM95 and CoALL-05-92. For patients with an adequate follow-up of 4 years the event-free survival of the 11q23/MLL-positive and 11q23/MLL-negative group was 0.2 or 0.64, respectively (P = 0.024). The monoclonal antibody 7.1. (moab 7.1) does not react with normal hematopoetic precursors or mature blood cells but was shown to specifically react with leukemic cells bearing a rearrangement of chromosome 11q23 or the MLL gene, respectively. We, therefore, specifically addressed the question whether the reactivity of moab 7.1, as determined by flow cytometry, may substitute for molecular testing of an 11q23/MLL rearrangement in this cohort of infant ALLs. Reactivity of moab 7.1 indicated a 11q23/MLL rearrangement with a specificity of 100%. However, five of the 11q23/MLL-positive cases did not react with moab 7.1 indicating a sensitivity of 84% only. Three of these five moab 7.1-negative but 11q23/MLL-positive cases could be identified by their unique expression pattern of CD65s and/or CD15. Thus, 95% of all 11q23/MLL-positive ALL cases in infancy may be identified by flow cytometry based on their expression of CD15, CD65s and/or moab 7.1.     

伴3'RARα亚显微缺失的急性早幼粒细胞白血病一例

 目的　报道1例伴RARα 3'-末端（3'RARα）亚显微缺失的M3r亚型急性早幼粒细胞白血病（APL）病例及其形态学、细胞遗传学、分子遗传学和分子生物学的研究结果。方法　骨髓细胞直接法和短期培养法制备染色体,应用反带技术进行核型分析;分别用CEP X／Y α-卫星DNA探针、LSI PML-RARα 双色双融合探针和LSI RARα 双色断裂点分离探针进行荧光原位杂交（FISH）分析;实时定量反转录聚合酶链反应（RT-PCR）方法检测PML-RARα 融合基因转录本;多重巢式RT-PCR技术检测急性白血病29种染色体畸变所形成的融合基因,包括PML-RARα,PLZF-RARα和NPM-RARα融合基因转录本。结果　反带分析显示核型为45,X,-Y[6]／46,XY[8],CEP X／Y探针FISH进一步证实了Y染色体丢失;RARα双色断裂点分离探针FISH分析显示1个RARα等位基因的整个3'-末端缺失;通过细胞遗传学、FISH以及RT-PCR等方法检测,排除PML-RARα、PLZF-RARα、NPM-RARα、NuMA-RARα和STAT5b-RARα重排。结论　识别APL中一种新的RARα 基因重排类型（3'RARα 亚显微缺失而无X-RARα 融合）,RARα 双色断裂点分离探针FISH分析是明确该异常的有效手段,其分子学结果有待进一步阐明。     

Genetic and clinical characterization of 45 acute leukemia patients with MLL gene rearrangements from a single institution

Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant.