间期荧光原位杂交检测核心结合因子相关急性白血病的初步研究

目的 应用间期荧光原位杂交技术(I-FISH)检测初诊急性白血病患者中核心结合因子(CBF)受累的染色体异常情况并对治疗后患者的微小残留病(MRD)进行监测，同时对I-FISH与常规染色体G显带的灵敏度进行比较。方法在骨髓形态学初筛的基础上，应用常规染色体G显带技术对15例急性髓系白血病(AML)进行核型分析，应用I-FISH对患者可能受累的CBF相关靶基因进行检测，其中7例患者治疗后进行了MRD监测。结果(1)正常对照组中，CYTOCELL公司提供的3种探针(AML1／ETO易位探针、MYH11基因断裂点双色探针和ETV6／AML1易位探针)的正常分界值分别为4．13％、1．95％和2．12％。(2)常规染色体G显带分析，15例患者中有6例伴有潜在累及CBF基因的染色体异常，其中8例AML-M2中5例伴t(18；21)，2例AML-M4EO中1例伴inv(16)，5例儿童B系急性淋巴细胞白血病(B—ALL)未检出相应的异常。I-FISH检测15例患者发现有12例累及CBF基因，其中8例AML-M2患者均为AML1/ETO融合基因( )，2例．AML-M4EO病人CBFμ／MYH11融合基因均为( )，5例儿童B-ALL，中2例ETV6／AML融合基因( )。(3)3例AML-M2患者中2例MRD阳性；2例M4EO患者治疗后MRD监测结果均阴性；2例B-ALL患者l例阴性m1例阳性。结论 I-FISH较常规染色体G显带技术能够更灵敏地检测出累及CBF的急性白血病。在初诊时联合应用这两种方法能使结果更趋全面和准确。     

染色体原位杂交检测28例急性早幼粒细胞白血病残留病变

目的:应用荧光染色体原位杂交(FISH)方法检测急性早幼粒细胞白血病(acutepromyelocyticleukemia,APL)患者完全缓解(completeremission,CR)期微小残留病变(minimalresidualdisease,MRD),并探讨MRD的动态变化与复发的关系。方法:对28例具有t(15;17)的APL患者在CR期应用中期分裂相FISH方法,进行了监测。结果:11例在CR的不同时期检出了不同比例的t(15;17)细胞,其中3例复发。17例在追踪的标本中均未检出异常细胞,目前均持续缓解。观察时间分别为10～33个月和15～44个月。结论:FISH追踪MRD较传统细胞遗传学更加快速和敏感,并具有可定量的优点,可作为APL患者CR期监测MRD的常规手段。FISH检测到MRD有增长趋势时,更有预示复发的价值。     

染色体原位杂交检测28例急性早幼粒细胞白血病残留病变

应用荧光染色体原位杂交（ＦＩＳＨ）方法检测急性早幼粒细胞白血病（ａｃｕｔｅｐｒｏｍｙｅｌｏｃｙｔｉｃｌｅｕｋｅｍｉａ,ＡＰＬ）患完全缓解（ｃｏｍｐｌｅｔｅｒｅｍｉｓｓｉｏｎ,ＣＲ）期微小残留病变（ｍｉｎｉｍａｌｒｅｓｉｄｕａｌｄｉｓｅａｓｅ,ＭＲＤ）,并探讨ＭＲＤ的动态变化与复发的关系。方法：对２８例具有ｔ）（１５：１７）的ＡＰＬ患在ＣＲ期应用中期分裂相ＦＩＳＨ方法,进行了监测。结果：１１例以     

荧光原位杂交技术在急性淋巴细胞白血病异常染色体检测中的应用

在急性淋巴细胞白血病（ＡＬＬ）患者中，荧光原位杂交（ＦＩＳＨ）的应用，可以更容易检测到异常１７、１８和２１号染色体，我们通过对３８例ＡＬＬ染色体检查，对荧光原位杂交及染色体Ｇ分带技术进行了比较。一、对象和方法１对象：１９９３年１２月～１９９５年２月...     

联合应用染色体G显带和间期荧光原位杂交技术检测急性早幼粒细胞白血病

目的 联合染色体G显带和间期荧光原位杂交技术(interphase fluorescence in situ hybridization，I—FISH)对急性早幼粒细胞白血病(acute promyelocytic leukemia，APL．)患者进行早期诊断和治疗后微小残留病的监测，同时对两种方法的灵敏度进行比较。方法 应用染色体G显带技术和I—FISH对20例APL患者进行遗传学检测，包括13例初诊患者和8例治疗后获完全缓解的患者(其中有初诊中的1例)。结果 ①13例初诊病例中，9例染色体G显带检测t(15；17)阳性，2例阴性,2例因分裂相缺乏无法分析结果，阳性检出率为9／13(69.2％)；I—FISH检测PML／RARa融合基因13例为阳性，阳性检出率为13／13(100％)，细胞阳性率为76％～100％，平均91.3％。②8例治疗后血液学获完全缓解病例，染色体G显带后核型分析6例正常，2例无分裂相可供分析；I—FISH检测7例均为阴性，1例阳性。结论 联合运用染色体G显带和I—FISH技术能够有效提高APL患者的诊断和治疗后微小残留病的监测水平。     

白血病微小残留病检测的临床研究进展

随着化疗、特异靶向治疗、造血干细胞移植技术的不断提高,白血病的治疗效果日益改善,然而复发仍是困扰白血病治愈的一个难点.     

荧光原位杂交在急性早幼粒细胞白血病治疗前后的研究

目的 探讨荧光原位杂交技术在急性早幼粒细胞白血病(APL)诊断及其微小残留病(MRD)检测中的价值。方法 应用PML/RARα易位探针对8例初诊和6例完全缓解的急性早幼粒细胞白血病和1例正常对照进行t(15；17)融合基因检测，并与经典细胞遗传学结果进行比较分析。结果 G显带分析8例初诊患者均为t(15；17)，6例缓解患者中3例为t(15；17)，3例为正常核型。FISH检测显示，8例初诊和5例缓解患者均存在PML／RARα融合基因红，绿荧光信号，只有1例缓解患者和正常对照为正常15红色信号和17绿色信号。研究发现2例M3V患者PML/RARα融合基因阳性率达80％以上，一般M3阳性率在60％左右，缓解中的患者阳性率普遍下降在20％以下，另外发现2例15三体患者和2例伴RML／RARα融合基因的同时增加1条17号染色体的绿色信号。结论 FISH技术是一种高灵敏度、高分辨率的分子遗传学新技术，对M3的诊断及缓解后残余的畸变肿瘤细胞检测具有重要意义。     

荧光原位杂交检测急性髓性白血病21号染色体复杂核型异常

目的对急性髓性白血病（AML）患者可能出现的21号染色体复杂核型异常进行研究。方法AML患者共50例,其中成人37例,儿童13例,采用荧光原位杂交技术（FISH）,运用多种位点特异性DNA探针（染色体全染、特殊位点、双色易位融合探针）进行杂交。结果50例AML中,7例患者出现21号染色体异常（14％）,包括21号染色体数量和结构上的异常。其中4例儿童AML出现21号染色体三倍体,1例合并复杂的核型变化：47～49,XX,der（1）t（1;17）（p36．1;q23）,＋4,＋10,der（11）t（11;17）（q23;q23）,-17,-18,＋20,＋21。3例成人AML出现21号染色体结构的变化,即t（8;21）（q22;q22）。其中1例患者出现复杂的核型变化,即der（21）,t（8;21）（q22;q22）,dup（15q）。结论AML常合并有21号染色体畸变。儿童及成人AML出现21号染色体畸变的方式不同：前者多见21号染色体数量上的变化,而后者多见21号染色体结构上的变化。     

组合探针荧光原位杂交检测慢性淋巴细胞白血病分子遗传学异常

目的 探讨中国人慢性淋巴细胞白血病(CLL)的分子遗传学特性及其在CLL预后判断中的价值.方法 运用组合探针(LSI D13S319、LSI p53、LSI ATM、CEP 12、LSI MYB、LSI IGHC/IGHV)和间期荧光原位杂交(FISH)技术,前瞻性研究106例CLL患者的的分子遗传学特征.生存分析采用Kaplan-Meier法绘制生存曲线和Log-rank检验.结果 106例患者中79例(74.5%)存在一种及以上细胞遗传学异常,35例(33.0%)同时检测出2种及以上的异常.其中del(13q14)异常最常见(45.3%),其他依次为+12(25.5%)、IgH基因重排(23.6%)、del(17p13,16.0%)、del(11q23,10.5%)和del(6q23,4.7%)异常.del(13q14)异常的发生率在年龄＜60岁的患者中明显高于≥60岁的患者,差异具有统计学意义(P=0.033);分子遗传学异常与患者性别、Binet分期无明显相关性(P＞0.05).生存分析显示,伴有del(17p13)或del(11q22)异常组患者的生存期最短,单纯具有del(13q14)异常组的生存期最长,差异具有统计学意义(P=0.003).结论 中国人CLL的分子遗传学特性与西方国家的相似,组合探针FISH技术提高了CLL异常染色体的检出率,del(13q14)异常是CLL最常见的染色体异常.     

荧光原位杂交联合染色体分析提高急性白血病的诊断率

目的:探讨荧光原位杂交(fluorescence in situ hybridization,FISH)检测联合染色体分析提高急性白血病(acute leukemia,AL)的临床诊断率。方法:无菌抽取2011年1月到2012年12月410例初诊为AL患儿的骨髓标本,经细胞培养后,采用G显带方法进行染色体分析,其中57例患儿用FISH技术进行相应的基因检测。结果:在410例AL患儿中,包括明确诊断为急性髓系白血病(acute myeloblastic leukemia,AML)132例,急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)251例,未明确诊断27例,染色体异常检出率为41%(168/410)。在57例AL患儿检测的染色体和FISH中,染色体异常检出率为52.6%(30/57);而FISH检测异常率为68.4%(39/57),两种方法检测的一致性为82.5%。结论:FISH方法是检测白血病患儿染色体异常的有效技术,可提高染色体异常检出率,明显优于单纯G显带染色体分析法,联合应用FISH及染色体分析对急性白血病的诊断结果更加准确、可靠,更适合于临床诊断、疗效判定。     

Chromosomal changes detected by fluorescence in situ hybridization in patients with acute lymphoblastic leukemia

Objectives To investigate patients with acute lymphoblastic leukemia (ALL) for TEL/AML1 fusion,BCR/ABL fusion, MLL gene rearrangements, and numerical changes of chromosomes 4, 10, 17 and 21 by fluorescence in situ hybridization (FISH) and to determine the relationship and the significance of those findings.Methods Fifty-one American patients (34 men and 17 women) were included in this study. Of them there were 41 patients with pro-B cell type ALL, 9 with B cell type ALL and 1 with T cell type ALL.Chromosome metaphases of each sample were prepared according to standard protocols.Fluorescence in situ hybridization was performed using commercially available DNA probes, including whole chromosome painting probes, locus specific probes, specific chromosome centromere probes and dual color/multiple color translocation fusion probes. The digital image analysis was carried out using Cytovision and Quips FISH programs.Results An overall incidence of chromosomal anomalies, including t (9; 22 ), MLL gene rearrangements, t (12;21), and numerical chromosomal anomalies of chromosomes 4, 10, 17 and 21 was found in 33 patients (65%). Thirty-one of them were pediatric patients and two adults. The t(12;21) was the commonest chromosomal anomaly detected in this population; 14 out of the 45pediatric patients (31%) were positive for TEL/AML1 fusion, among which three had an additionalderivative 21[t (12;21) ], four had a deletion of 12p and two had an extra copy of chromosome 21.All 14 patients with positive TEL/AML1 fusion had ALL pre-B cell or B-cell lineage according to standard immunotyping. The percentage of cells with fusion signals ranged from 20% to 80%. All fourteen patients positive for TEL/AML1 gene fusion were mosaic. Three out of the 14 patients positive for the TEL/AML1 gene fusion were originally reported to be culture failures and none of the remaining eleven samples had been found to have chromosome 12 abnormalities by conventional cytogenetic techniques. All pediatric patients with pre-T or T cell lineage and the six adults were negative for TEL/AML1 fusion. One patient had double Philadelphia chromosomes, three had a rearranaement or a deletion of the MLL aene. one had t (4;11)and two had a deletion of the MLL One of the patients with an MLL deletion also had a large ring of chromosome 21, and r (21) was caused by AML1 gene tandemly duplicated at least five times. The second case with the MLL deletion was also unique, the patient had a t (12;21) as well. A total of 20 patients had numerical changes( gain or loss) of chromosomes 4, 10, 17 and 21. Eight patients were found to have trisomies of three or four different chromosomes. Interestingly, seven of these patients did not have TEL/AML1, BCR/ABL or the MLL aene rearranaement, one did have the TEL/AML1 aene fusion. Eleven patients with pro-B cell or B cell type ALL (9 children with ALL, 2 adults with ALL) had numerical changes of chromosome 21 (gain 1 or 2 chromosome 21 ), among them, 10 patients had no structural alteration of chromosome 21, and one was combined by t (12;21 ). Four patients had a monosomy of chromosome 17 and three out of these patients with monosomy 17 also had a fusion signal of TEL/AML1.     

优化的预包被式多探针荧光原位杂交技术可提高白血病细胞遗传学异常的检测效率

目的探讨对预包被式多探针荧光原位杂交(FISH)技术进行优化在检测白血病细胞遗传学异常的意义。方法 141例初诊为ALL或AML/MDS的患者骨髓标本,行预优化的包被式多探针FISH流程检测(在原始流程的基础上增加了调整细胞密度、蛋白酶消化步骤),其中35例行常规流程检测,对比优化前后对白血病细胞遗传学异常的检测成功率与阳性位点检出率。结果对多探针FISH实验流程进行优化后,ALL的检测成功率由85.3%提高到100%,阳性位点检出率由5.1%提高到8.6%;AML/MDS的检测成功率由67.4%提高到99.8%,阳性位点检出率由3.5%提高到6.0%,差异均具有统计学意义(P<0.01)。结论通过对常规预包被式多探针FISH系统检测流程进行优化,显著提高了白血病细胞遗传学异常的检测效率,增强了该技术的临床实用性。     

双色双融合间期荧光原位杂交探针检测慢性髓系白血病伊马替尼治疗患者的肿瘤负荷

目的研究双色双融合探针间期荧光原位杂交技术(D-FISH)检测慢性髓系白血病(CML)伊马替尼治疗过程中肿瘤负荷的灵敏度、特异性。方法应用D-FISH探针对24例伊马替尼治疗获完全细胞遗传学缓解的CML患者骨髓内残留肿瘤细胞负荷进行检测,并与应用单融合探针FISH(S-FISH)和逆转录-聚合酶链反应(RT-PCR)检测结果的进行比较。结果与S-FISH相比,D-FISH较具有更高的灵敏度及特异性。正常对照组中D-FISH的正常分界值为0.73%,而S-FISH为6.24%,差异具有显著性;在24例伊马替尼治疗后患者中,S-FISH检测到7例(29.2%)阳性,而D-FISH检测到13例(54.2%)阳性,且结果与RT-PCR结果高度相关。结论D-FISH由于具有较低的假阳性率与假阴性率,可较灵敏、特异地监测CML伊马替尼治疗过程中肿瘤细胞负荷的变化。     

间期荧光原位杂交结合测序分析在慢性髓系白血病慢性期伊马替尼治疗监测中的应用

目的:探讨在慢性髓系白血病(CML)慢性期伊马替尼治疗中,间期荧光原位杂交(I-FISH)结合Abl酪氨酸激酶区测序技术对疗效监测、耐药分析及预后判断的意义.方法:应用双色双融合探针I-FISH对18例伊马替尼治疗获完全细胞遗传学缓解的CML慢性期患者骨髓内残留肿瘤细胞负荷进行检测,并对I-FISH检测阳性患者进行ABL酪氨酸激酶区测序分析.结果:双色双融合探针I-FISH的正常分界值为0.73%,18例伊马替尼治疗后患者检测中,8/18(44%)结果阳性;8例I-FISH检测阳性患者测序分析发现2例(25%)出现ABL激酶区点突变,点突变类型为E255K和T315I.结论:联合应用I-FISH和测序分析不仅能较灵敏、特异地监测伊马替尼治疗后肿瘤细胞负荷的变化,并可对药物疗效、耐药情况进行早期分析,有效地指导治疗和判断预后.     

八探针间期荧光原位杂交技术联合染色体核型分析比较成人与儿童急性淋巴细胞白血病患者的细胞遗传学差异

目的应用八探针间期荧光原位杂交技术(FISH)联合染色体核型分析观察急性淋巴细胞白血病(ALL)成人患者与儿童患者的细胞遗传学差异。方法对125例ALL患者(成人86例、儿童39例)全部行八探针FISH(MYC、P16、E2A、TEL/AML1、BCR/ABL、MLL、IGH、多倍体的DNA探针)检测并染色体核型分析。结果八探针FISH检测结果显示,成人ALL患者与儿童ALL患者的TEL/AML1融合基因、BCR/ABL融合基因与多倍体阳性率之间的差异有统计学意义(P<0.05);染色体核型分析成人ALL患者与儿童ALL患者的(t9;22)易位、多倍体阳性率之间的差异有统计学意义(P<0.05)。结论成人ALL患者与儿童ALL患者融合基因表达各有侧重,不同的细胞遗传学特征与其预后密切相关。FISH多探针诊断系统检测ALL患者常见遗传学异常省时、准确、高效,与染色体核型分析形成很好的互补。     

慢性髓细胞白血病的细胞遗传学和荧光原位杂交分析

目的 探讨慢性髓细胞白血病（chronic myeloid leukemia,CML）细胞遗传学、分子遗传学特点及其临床意义.方法 选择196例CML患者,采用直接法和（或）24 h短期培养法制备骨髓染色体标本,采用R显带技术（RHG）进行染色体核型分析;用BCR-ABL双色双融合探针或ES探针对骨髓间期细胞进行荧光原位杂交（fluorescence in situ hybridization,FISH）检测.结果 将患者分为慢性期和加速/急变期2组.细胞遗传学分析：附加染色体异常、变异易位在2组中比例分别为9/166（5.4%）、7/166（4.2%）和19/30（63.3%）、3/30（10.0%）.196例患者中检出179例Ph阳性标本,占91.3%;其中典型易位169例（94.4%）,变异易位10例（5.6%）,附加染色体异常出现频率最多的为双Ph、＋ 8、I（17q）、-Y.分子遗传学分析：68例患者行FISH 检测,均为BCR-ABL融合基因阳性（含10例变异易位、11例Ph染色体阴性及6例染色体检测失败者）,其中10例（14.7%）为der（9）缺失（含1例染色体检测失败者）,在慢性期、加速/急变期中比例分别为8/55（14.5%）、2/13（15.4%）.68例患者中典型易位41例,变异易位10例,der（9）缺失在典型易位、变异易位中比例分别为5/41（12.2%）和4/10（40.0%）.结论 与慢性期相比,加速/急变期附加染色体、变异易位等异常明显增加,染色体核型分析有助于疾病诊断和进展的分析,并且在遗传学分析的基础上,选用分子遗传学FISH技术可以明显提高疾病的诊断率和染色体异常的检出率及准确率,并可有效检测der（9）缺失.     

Cytogenetic characteristics of B cell chronic lymphocytic leukemia in 275 Chinese patients by fluorescence in situ hybridization: a multicenter study

Background  Under conventional cytogenetic (CC) analysis, only 30%–50% of B cell chronic lymphocytic leukemia (B-CLL) cases show clonal aberrations. Using fluorescence in situ hybridization (FISH), the percentage of patients with abnormalities rises to almost 80%, among them, the most frequent abnormalities were 13q14, 11q22, p53 deletions and trisomy 12. The aim of this study was to explore the incidence of cytogenetic changes in Chinese patients with B-CLL.

Methods  We used FISH methods to detect the cytogenetic features in 275 cases of B-CLL from 48 hospitals. The correlation between FISH abnormalities and clinical characteristics such as age, gender, white blood cell count, peripheral hemoglobin (Hb) level, peripheral platelet count (PLT), lactate dehydrogenase (LDH) level, Rai stage, Binet stage, and overall survival was analyzed, and the relationship between them and overall survival was also analyzed to evaluate their prognostic implications.

Results  Of the 275 patients, genetic aberrations were found in 77.8% using FISH. The frequencies of abnormalities were as follows: 13q deletion (56.4%), trisomy 12 (34.5%), p53 deletion (33.5%) and 11q22 deletion (30.5%). It was obvious that the patients with p53 deletion had lower level of Hb (P=0.001) and PLT (P=0.003) when compared to patients without p53 deletion. Significant differences were obtained in the distribution of p53 deletion according to Rai and Binet classification systems (P=0.016 and 0.008 respectively). Significant differences were also observed when the overall survival was correlated with p53 deletion (P=0.043), Rai stage (P=0.006), Binet stage (P=0.013), Hb level (P=0.004) and PLT level (P=0.010).

Conclusions  Chinese CLL patients have the similar frequencies of del(13q), trisomy 12, del(11q) and a higher frequency of del(17p) when compared to literatures. Del(17p) is associated with advanced stage and low levels of Hb and PLT. Patients with p53 deletion, or advanced stage probably have poor survival in China.