FISH联合multiplex RT-PCR检测MLL基因重排的价值探讨

目的:采用荧光原位杂交(FISH)与逆转录多重巢式聚合酶链反应(multiplexRT-PCR)技术检测急性白血病中MLL基因重排的情况,分析两者联合应用的诊断价值。方法:对2008年1月~2011年5月在我院诊断为急性白血病的201例患者采用MLL双色断裂分离重排探针进行FISH检测,同时用multiplexRT-PCR技术检测11种较常见的MLL融合基因,观察MLL基因异常的检出率。所有患者均进行常规染色体核型分析(CCA),观察11q23重排率作为对照。结果:共有19例患者出现11q23／MLL基因重排,在急性髓细胞白血病(AML)中的检出13例(10.2%),急性淋巴细胞白血病(ALL)的检出6例(8.2%)。FISH联合multiplexRT-PCR对MLL基因重排的检出率为9.45%,CCA对11q23异常的检出率为5.47%。5例正常核型的患者和3例未涉及11号染色体异常的患者中FISH检出了1例MLL倒位和3例扩增信号,multiplexRT-PCR检出了7例dupMLL(11q23)重排。结论:FISH联合multiplexRT-PCR能提高MLL基因重排检出率。     

八探针荧光原位杂交联合R显带技术诊断儿童急性髓系白血病CSCD

目的探讨将八探针荧光原位杂交（fluorescence in situ hybridization, FISH）联合R显带染色体核型分析应用于儿童急性髓系细胞白血病（acute mydoid leukemia,AML）诊断的价值。方法应用八探针FISH（AML1／ETO、PML—RARa、CBFβ／MYH11、mL、P53、5q-、7／7q-、20q-等8种DNA探针）和R显带染色体核型分析技术,对214例AML患儿进行了联合检测。结果八探针FISH技术在118例患儿中检出了细胞遗传学改变,总体阳性率为55．1％,包括AML1／ETO、PML／RARa、CBFβ／MYH11、MLL、P53、5q-、7／7q-、20q-等8种细胞遗传学异常。R显带核型分析检出染色体异常55例,阳性率为25．7％,其中4例染色体异常FISH未检出。两种方法检出阳性率的差异有统计学意义（P〈0．05）。结论八探针FISH技术较R显带染色体核型分析具有准确、高效、省时、省力等优点,可与染色体核型分析有效互补,并且每种细胞遗传学异常都可为儿童AML的诊断、预后评估和个体化治疗提供重要依据。     

30例急性髓细胞性白血病细胞遗传学特征

目的对急性髓细胞性白血病(AML)患者的细胞遗传学特征进行调查和分析,探讨FISH在儿童AML临床诊断中的应用。方法回顾性分析河北省儿童医院2011年4月至2013年10月收治的30例AML患儿的FISH及染色体核型结果。结果 1.染色体核型G、R显带方法:分析30例样本,检出18例正常核型,2种(3例)单纯数量异常,2种(6例)单纯结构异常,1例结构伴数量混合异常,2例无分裂相。2.FISH方法:30例AML,12例正常核型,18例结果阳性,包括AML/ETO阳性8例,CBFβ阳性5例,MLL重排2例(1例合并+8),1例21×2,1例+X,+1,+14,1例8+,+21,-Y。22例两种方法检出一致,其中正常核型14例,t(8;21)即AML/ETO阳性4例,inv(16)即CBFβ1例,MLL重排1例,1例21*2,1例+X,+1,+14。结论 FISH与染色体显带技术两种方法具有一致性,FISH异常检出率高于显带技术,且存在统计学差异。FISH技术能够使遗传学异常的检测更准确,在儿童AML的诊断和分型中具有值得推广的临床价值。     

联合间期和中期荧光原位杂交检测发现11q23异常伴D13S319缺失急性髓系白血病一例

目的对1例出现11q23异常伴D13S319缺失的罕见急性髓系白血病(acute myeloid leukemia,AML)患者进行多途径细胞遗传学分析,为诊断、治疗及预后分层提供依据。方法应用G+R显带技术对患者24 h培养后的中期分裂相进行染色体核型分析,联合分裂间期和中期荧光原位杂交(fluorescence in situ hybridization,FISH)技术对患者染色体的特定位点进行检测,明确复杂易位和微小缺失片段。结果患者存在混合系白血病基因(mixed lineage leukemia,MLL)重排,形成了MLL-AF10融合基因,并伴有13号染色体D13S319位点的缺失。结论MLL基因重排合并D13S319位点缺失在急性白血病中是否具有双重打击效应应引起临床的重视。在AML患者核型中发现13q-、del(13)(q14)、-13或der(13)任意一种克隆性异常时,应行FISH检测论证及明确缺失片段的大小,以便进行靶向治疗。     

伴有11q23/MLL重排的儿童急性髓系白血病的临床及实验室分析

目的 分析伴有11q23/混合谱系白血病(mixed lineage leukemia,MLL)基因重排的儿童急性體系白血病(acute myeloid leukemia,AML)的临床和实验室特点.方法 采用骨髓细胞短期培养法和R显带技术对234例初诊AML患儿进行核型分析;采用逆转录-多重巢式聚合酶链反应(多重PCR)技术检测MLL融合基因以及MLL部分串联重复;采用双色MLL基因探针,对其中2例核型分析具有11q23易位而多重PCR检测MLL融合基因呈阴性的患儿样本进行间期双色荧光原位杂交(dual-color fluorescencein situ hybridization,D-FISH)MLL重排检测.结果 R显带提示234例初诊AML患儿中,20例(M5 14例、M4 4例、M2 2例)有涉及11q23的易位,包括t(9;11)(p22;q23)12例,t(1;11)(q21;q23)3例,t(6;11)(q27;q23)2例,t(11;19)(q23;p13)、t(5;11)(q31;q23)和t(X;11)(q24;q23)各1例.多重PCR证实其中18例有MLL的融合转录本,有2例阴性,但D-FISH均检出MLL重排;在其余AML患儿的样本中检出8例(M5 4例、M4 2例、M2和M6各1例)有MLL部分串联重复.本组AML患儿中,11q23/MLL重排的总检出率11.97%(28/234),其中85.7%(24/28)的病例为M4/M5亚型.本组28例伴有11q23/MLL重排患儿,治疗后完全缓解率为53.8%,与对照组(以同期伴有其他异常核型和正常核型的AML-M4/M5患儿共27例作为对照)的90.5%相比,差异有统计学意义(P<0.05).其中2例患儿接受了强烈化疗,分别生存达81和66个月.4例接受了异基因干细胞移植,已分别生存21、20、16和11个月,至今仍在完全缓解中.本组28例伴有11q23/MLL重排患儿的中位生存期为11个月,对照组为15个月,差异无统计学意义(P>0.05).结论 伴有11q23/MLL重排的AML患儿和单核系白血病高度相关.11q23易位和MLL部分串联重复是相互排斥的,二者预后均较差.采用强烈化疗和异基因干细胞移植有望获得较好疗效.多重PCR联合染色体核型分析和D-FISH技术是对初诊AML患者进行各种MLL重排筛检的有效方法.     

38例儿童急性淋巴细胞白血病染色体结果分析

目的对儿童急性淋巴细胞白血病(ALL)中的染色体核型及融合基因进行分析。方法应用骨髓染色体标本及FISH方法对38例儿童ALL患者做染色体核型分析及融合基因检测。结果 38例检测患儿中发现染色体异常核型7例;融合基因检测阳性者12例,其中7例是TEL/AML1阳性。结论细胞遗传学联合融合基因检测有助于儿童ALL的诊断及预后判断分析。     

急性单核系白血病M4/M5中MLL基因重排的间期荧光原位杂交研究

目的　探讨间期荧光原位杂交 ( fluorescence in situ hybridization,FISH)技术对混合谱系白血病 ( mixed lineage leukemia,ML L)基因重排检测的价值 ;评估 ML L 重排在急性单核系白血病 M4 / M5中的发生率和预后意义。方法　采用骨髓直接法或短期培养法制备染色体 ,应用 R显带技术进行核型分析。采用地高辛标记的跨越 11q2 3断裂位点的单色 ML L探针和间期 FISH技术对 2 3例急性单核系白血病M4 / M5病例进行 ML L重排检测。结果　 R显带揭示 2 3例中 7例有涉及 11q2 3的易位 ,5例有其他核型异常 ,10例核型正常 ,1例核型分析失败。 FISH研究显示 12例有 ML L重排 ,包括 R显带检出 11q2 3异常的7例。结论 FISH技术检测 ML L重排的敏感性明显高于常规细胞遗传学技术 ;ML L重排与急性单核系白血病 M4 / M5高度相关 ,是预后不良的指标     

应用光谱染色体核型分析技术研究胰腺癌细胞系P2染色体核型

目的 探讨胰腺癌的细胞遗传学特征.方法 采用光谱核型分析技术对中国人胰腺癌细胞系P2的染色体核型进行分析,并选择EGFR/CEP 7双色荧光原位杂交(FISH)探针,对比分析10例胰腺癌和10例慢性胰腺炎石蜡标本的EGFR基因拷贝数,验证光谱核型分析结果.结果 P2细胞系为亚三倍体核型,共发现26种染色体异常,其中重复出现的染色体异常改变为染色体4、9、18、19、22、Y、10p、15p、8p、6q和12p缺失,染色体7和12q增加,以及染色体结构畸变der(9;15)(q10;q10)、der(10)(3;10)(?;q26)和der(12)(8;12)(?;p13).EGFR-FISH阳性为4/10.结论 胰腺癌细胞系的染色体重排非常复杂,进一步扩大样本量进行相关分析,包括了解胰腺癌的EGFR-FISH阳性率非常有必要.     

应用光谱染色体核型分析技术研究胰腺癌细胞系P2染色体核型

目的 探讨胰腺癌的细胞遗传学特征.方法 采用光谱核型分析技术对中国人胰腺癌细胞系P2的染色体核型进行分析,并选择EGFR/CEP 7双色荧光原位杂交(FISH)探针,对比分析10例胰腺癌和10例慢性胰腺炎石蜡标本的EGFR基因拷贝数,验证光谱核型分析结果.结果 P2细胞系为亚三倍体核型,共发现26种染色体异常,其中重复出现的染色体异常改变为染色体4、9、18、19、22、Y、10p、15p、8p、6q和12p缺失,染色体7和12q增加,以及染色体结构畸变der(9;15)(q10;q10)、der(10)(3;10)(?;q26)和der(12)(8;12)(?;p13).EGFR-FISH阳性为4/10.结论 胰腺癌细胞系的染色体重排非常复杂,进一步扩大样本量进行相关分析,包括了解胰腺癌的EGFR-FISH阳性率非常有必要.     

一例伴t(14;14)(q11;q32)易位的B细胞急性淋巴细胞白血病的临床和分子细胞遗传学研究

目的 报告1例伴t(14;14)(q11;q32)易位的罕见B细胞急性淋巴细胞白血病(B-lineage acute lymphoblastie leukemia,B-ALL)病例,阐明其临床和分子细胞遗传学特征.方法 分析1例伴t(14;14)(q11;q32)易位B-ALL患者的临床资料;将患者骨髓细胞24h培养后按常规方法制备染色体标本,采用R显带技术进行核型分析;分别应用IGH双色断裂点分离探针、CEBPE双色断裂点分离探针、4号全染色体涂染探针和ALL组合探针进行荧光原位杂交(fluorescence in situ hybridization,FISH)分析.结果 常规细胞遗传学分析显示患者核型为47,XX,+4,t(14;14)(q11;q32)[20],FISH分析进一步证实了这种核型异常.IGH双色断裂点分离探针FISH分析表明t(14;14)(q11;q32)易位累及IGH基因,CEBPE双色断裂点分离探针FISH分析提示t(14;14)(q11;q32)易位中IGH的伙伴基因为CEBPE基因.结论 在B-ALL中t(14;4)(q11;q32)易位同时累及IGH和CEBPE基因为少见的再现性遗传学异常,该异常可定义B-ALL中一种新的亚型.伴有t(14;14)(q11;q32) IGH/CEBPE易位的B-ALL患者可能预后较好.     

Spectra of chromosomal aberrations in 325 leukemia patients and implications for the development of new molecular detection systems

This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.     

The value of fluorescence in situ hybridization in the diagnosis and prognosis of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. Prognosis is related to clinical staging and cytogenetic findings. Conventional cytogenetic analysis of CLL reveals abnormalities in approximately one third of patients. Fluorescence in situ hybridization (FISH) is analytically more sensitive than conventional cytogenetics for specific chromosomal abnormalities. To evaluate the usefulness of FISH in CLL, a study of 100 CLL patients comparing conventional cytogenetics and a commercially available multiprobe FISH kit was undertaken. One hundred consecutive CLL patients (67 males, 33 females) were studied. The male-female patient ratio was approximately 2.0 to 1. Twenty-eight percent (28/98) of patients had abnormal karyotypes by conventional cytogenetics (one patient had no specimen for conventional cytogenetics and one had an unanalyzable karyotype), and of those 19/100 (19%) had more than one chromosomal abnormality. Sixty-four percent (64/100) of the patients were positive for at least one abnormality by the FISH probes used. The following abnormalities were noted with FISH: 11q22 ATM, 23/100 (23%); trisomy 12, 11/100 (11%); 13q14.3, 40/100 (40%); 13q34.3, 4/100 (4%); 17p13.1, 12/100 (12%). Conventional karyotypes revealed 2 patients with abnormalities of chromosome 6 (which FISH did not address); 11 with abnormalities of 11 or 11q; 6 with trisomy 12; and 4 with abnormalities of 17. Aberrations of 11q and 17p are reported to have a poor prognosis in CLL. FISH can identify abnormalities missed with conventional cytogenetics and is helpful in diagnosis, prognosis, and evaluation of therapy for CLL. Additional chromosomal changes are identified with conventional cytogenetics that are not addressed by the multiprobe FISH kit.     

11q23 abnormalities in patients with acute myelogenous leukemia and myelodysplastic syndrome as detected by molecular and cytogenetic analyses

11q23 chromosomal abnormalities and rearrangement of the mixed lineage leukemia (MLL) gene are important prognostic factors in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). However, the presence of 11q23 abnormalities does not always correlate with that of MLL gene rearrangement. We retrospectively compared the occurrence of 11q23 abnormalities (measured by karyotyping) and MLL gene rearrangement (measured by Southern blotting) in bone marrow from 311 consecutive adult patients with AML or MDS. 11q23 abnormalities were found in 18 patients (5.8%), of whom 7 (39%) did not have the MLL gene rearrangement. MLL gene rearrangement was detected in 35 patients (11.2%). Of these 35 patients, only 11 (31%) had cytogenetic evidence of 11q23 abnormalities. None of the 21 patients with chronic myelomonocytic leukemia had 11q23 abnormalities or MLL gene rearrangement. 11q23 abnormalities were associated with shorter survival than was a diploid karyotype. Both cytogenetic and molecular studies should be performed to detect 11q23 abnormalities in patients with AML or MDS.     

Identification of ins(8;21) with AML1/ETO fusion in acute myelogenous leukemia M2 by molecular cytogenetics

A high percentage of cases of acute myelogenous leukemia (AML) of the M2 subtype show a rearrangement between the AML1 and ETO genes. The detection of the AML1/ETO fusion has clinical relevance because patients with this subtype have a good prognosis. We present the results of conventional and molecular cytogenetic studies in a patient with acute myelogenous leukemia French-American-British M2 classification, who had a complex karyotype involving chromosomes 8 and 21. Dual-color fluorescence in situ hybridization (FISH) using the AML1/ETO probe demonstrated a recombination of both genes on an add(8) chromosome. The use of other FISH probes (CEP8, c-myc and TEL21) and spectral karyotyping indicated that AML1/ETO fusion occurred as a consequence of a previously undescribed ins(8;21)(q22;q22.1q22.3).     

Cryptic ins(4;11)(q21;q23q23) detected by fluorescence in situ hybridization: a variant of t(4;11)(q21;q23) in an infant with a precursor B-cell acute lymphoblastic leukemia report of a second case

We report the chromosomal findings in a 4-year-old female with precursor B-cell acute lymphoblastic leukemia (ALL). The diagnostic karyotype showed an isochromosome 7q, i(7)(q10), as well as questionable rearrangements on 9p and 11q. Fluorescence in situ hybridization (FISH) studies on both interphase and metaphase cells using the MLL "break-apart" and the centromeric chromosome 4 probes were instrumental in the characterization of an MLL gene rearrangement, which was cryptic by conventional cytogenetic analysis. Specifically, the FISH pattern was consistent with an insertion of the 5' region of the MLL gene into chromosome 4 at band q21, most likely a variant t(4;11)(q21;q23). This is the second case of FISH detection of an ins(4;11) in ALL. Our case exemplifies the importance of FISH in the further characterization of precursor B-cell ALL cases without any apparent prognostically significant chromosomal abnormalities.     

Cytogenetic profile in de novo acute myeloid leukemia with FAB subtypes M0, M1, and M2: a study based on 652 cases analyzed with morphology, cytogenetics, and fluorescence in situ hybridization

In about 55% of acute myeloid leukemia (AML) cases, chromosome aberrations are detectable by cytogenetics. Close correlations between cytomorphology and cytogenetics have been reported. To determine a pattern of cytogenetic abnormalities within the French-American-British (FAB) subtypes AML M0, M1, and M2, we analyzed 48 AML M0, 179 AML M1, and 425 AML M2 and compared cytogenetic data to a cohort of 1,062 AML M3/3v, M4, M4eo, M5a/5b, M6, and M7. Cytogenetic abnormalities were significantly more frequent in AML M0 (71%) compared to M1 (49%), M2 (53%), and the total cohort (56%; P < 0.02). While +8 was the most common numeric abnormality in all FAB subtypes, +13, +14, and +11 were associated with AML M0-M2. The only recurring balanced translocation that was associated with one of these FAB subtypes was t(8;21) in M2 (12.5%) and, rarely, M1 (1.7%) (M0, 0% and M3-7, 0.09%; P=0.001). To evaluate the frequency of cytogenetically undetectable abnormalities, we performed fluorescence in situ hybridization (FISH) analyses in 273 AML M0-M2 with normal karyotype using probes for ETO, ABL, MLL, TEL, RB, P53, AML1, and BCR. In two cases we identified numerical aberrations of RB only in interphases nuclei. In seven additional cases, TEL and MLL abnormalities were found. In conclusion, t(8;21), +11, +13, and +14 are strongly associated with AML M0, M1, and M2. The FISH screening analyses identified abnormalities in an additional 3% in normal karyotypes.