Reid Alistair G, Tarpey Patrick S, Nacheva Ellie P
Department of Academic Haematology, Royal Free and University College School of Medicine, University College London, United Kingdom.
Genes Chromosomes Cancer. 2003 Jul;37(3):282-90. doi: 10.1002/gcc.10215.
Chronic myeloid leukemia (CML) is a biphasic hematopoietic malignancy associated with a single cytogenetic aberration, the Philadelphia translocation t(9;22)(q34;q11), resulting in the BCR-ABL1 fusion oncogene. Molecular heterogeneity was recently demonstrated in the form of extensive deletion of chromosomes 9 and 22 material from the der(9)t(9;22) in 15% of CML patients. The deletions were associated with a worse disease prognosis. Further genetic heterogeneity is seen during the terminal blast crisis stage of CML, in the form of additional non-random chromosome abnormalities. These include most frequently an extra copy of the Ph chromosome, trisomy 8, and isochromosome 17q. We used the genetic heterogeneity of CML as a framework to explore a new technique for high-throughput assessment of locus copy number in malignancy. Multiplex amplifiable probe hybridization (MAPH) relies on the ability of numerous short (100-300 bp) DNA probes to be recovered quantitatively by use of a common primer pair after hybridization to genomic DNA. Derivative chromosome 9 deletions were successfully mapped in a CML cell line (MC3) and nine patient bone marrow samples by simultaneous hybridization of 10 MAPH probes. All results were confirmed by fluorescence in situ hybridization. MAPH was found to be informative in the presence of up to 50% of normal cells, thus establishing the sensitivity of the technique in clonal tumor cell populations. MAPH was performed effectively on DNA samples extracted from fresh or methanol/acetic acid-fixed clonal cell populations. Amplifications of BCR-ABL1 were also detected and quantified in four CML cell lines by use of MAPH probes specific for ABL1 exon 11 and BCR exon 1. Our results demonstrate that MAPH is a reproducible high-throughput method suitable for the assessment of genomic imbalances of multiple loci in tumor DNA samples with heterogeneous cell populations at a resolution of 100-300 bp.
慢性髓性白血病(CML)是一种双相造血恶性肿瘤,与单一细胞遗传学畸变——费城染色体易位t(9;22)(q34;q11)相关,该易位导致BCR-ABL1融合致癌基因的产生。最近在15%的CML患者中发现了分子异质性,表现为der(9)t(9;22)上9号和22号染色体物质的广泛缺失。这些缺失与更差的疾病预后相关。在CML的终末期原始细胞危象阶段还可见到进一步的遗传异质性,表现为额外的非随机染色体异常。其中最常见的包括费城染色体额外拷贝、8号染色体三体和17号染色体长臂等臂染色体。我们以CML的遗传异质性为框架,探索一种用于恶性肿瘤中基因座拷贝数高通量评估的新技术。多重可扩增探针杂交(MAPH)依赖于众多短(100 - 300 bp)DNA探针在与基因组DNA杂交后通过使用一对通用引物进行定量回收的能力。通过同时杂交10个MAPH探针,成功在一个CML细胞系(MC3)和9例患者骨髓样本中定位了9号衍生染色体缺失。所有结果均通过荧光原位杂交得到证实。发现MAPH在存在高达50%正常细胞的情况下仍具有信息价值,从而确立了该技术在克隆性肿瘤细胞群体中的敏感性。MAPH对从新鲜或甲醇/乙酸固定的克隆细胞群体中提取的DNA样本有效。还通过使用针对ABL1外显子11和BCR外显子1的MAPH探针,在4个CML细胞系中检测并定量了BCR-ABL1的扩增。我们的结果表明,MAPH是一种可重复的高通量方法,适用于评估具有异质细胞群体的肿瘤DNA样本中多个基因座的基因组失衡,分辨率为100 - 300 bp。