Cytogenetic Studies of B-cell Chronic Lymphocytic Leukemia (B-CLL)

在用多克隆B细胞有丝分裂原进行适当的体外刺激后，先前的G显带核型分析在40- 100%的B-CLL患者中检测到克隆染色体异常。尚不清楚不同系列中报告的异常频率的广泛变化是由于疾病特征的真正差异还是仅仅由于体外培养条件。常见的复发性染色体异常包括12三体、14 q32重排、13 q易位或缺失、6 q缺失和11 q缺失。最近的研究表明，增加检测三体12，13 q缺失，11 q缺失，和17 p（p53）缺失间期荧光原位杂交（FISH）技术，表明这些变化确实发生在病程的早期。关于细胞遗传学在B-CLL中的临床和预后意义的数据正在出现。这些数据表明某些核型和/或FISH异常与B-CLL的特定临床病理亚群以及疾病的病程相关。为了解决B-CLL核型变化的时间问题，以及这些变化的可能特异性和意义，我们一直在对NCI和NHLBI的散发性或家族性B-CLL患者进行前瞻性研究，以进行评估和可能的治疗。本项目的细胞遗传学具体目标是：确定从B-CLL患者外周血中获得核型异常有丝分裂细胞的最佳培养条件;确定间期FISH是否检测到被G带中期分析遗漏的克隆异常细胞;比较基因组杂交（CGH）将检测中期分裂相或FISH分析未发现的染色体物质的增加或丢失;并将细胞遗传学结果与疾病的临床、形态学和免疫表型特征相关联，用基因表达的cDNA微阵列分析，以及新疗法的效果。迄今为止，已有超过275例患者入组研究。我们从1997年到2004年的初步分析显示，使用G显带中期分析，克隆染色体异常的病例仅占30- 40%，并证明了e.大肠杆菌脂多糖作为白血病细胞的有丝分裂原。间期荧光原位杂交与面板的五个探针证实或扩大了G-带的发现异常克隆的患者，并已检测到异常，但约10%的患者测试的日期。从2002年到2005年，我们进行了CGH回顾性DNA分离的患者先前进入研究，并前瞻性DNA从新的患者。CGH在检测亚显微缺失方面不如间期FISH敏感，但检测到FISH未探测到的区域的增益和损失。结合G显带、FISH和CGH，我们能够在90%以上的B-CLL患者中发现分子细胞遗传学异常; FISH明显上级其他方法。此外，用两种探针的双重杂交以及在多个患者中的连续分析已经显示在诊断时存在某些异常，并且在转化时存在额外的异常。基于这些研究，我们现在对所有患者常规进行间期FISH。我们还在对新探针进行质量控制，以添加到常规FISH面板中。只有在特别指出的情况下才进行完整的G显带核型分析。间期FISH结果目前被用于根据风险对患者进行分类，并分配给治疗方案。细胞遗传学与诊断和转化时的临床和其他实验室特征以及与基因表达和对治疗的反应的相关研究正在进行中。

Previous G-banded karyotype analyses detected clonal chromosome abnormalities in 40-100 percent of B-CLL patients studied after appropriate in-vitro stimulation with polyclonal B-cell mitogens. It was unknown whether the wide variations in frequencies of abnormalities reported in different series were due to true differences in disease characteristics or merely to in-vitro culture conditions. Common recurring chromosomal abnormalities included trisomy 12, rearrangements of 14q32, translocations or deletions of 13q, deletions of 6q, and deletions of 11q. Recent studies have shown increased detection of trisomy 12, 13q deletions, 11q deletions, and 17p (p53) deletions with interphase fluorescence in-situ hybridization (FISH) techniques, suggesting these changes do indeed occur early in the course of the disease. Data regarding the clinical and prognostic significance of cytogenetics in B-CLL are emerging. The data suggest certain karyotypic and/or FISH abnormalities are associated with specific clinicopathologic subsets of B-CLL, and with the course of the disease. To address the questions of timing of karyotypic changes in B-CLL, and the possible specificity and significance of these changes, we have been conducting prospective studies of patients with sporadic or familial B-CLL referred to the NCI and NHLBI for evaluation and possible treatment. The cytogenetics specific aims of this project are: to determine the optimal culture conditions for obtaining karyotypically abnormal mitotic cells from peripheral blood of patients with B-CLL; to determine whether or not interphase FISH detects clonally abnormal cells missed by G-banded metaphase analysis; to determine whether or not comparative genomic hybridization (CGH) will detect gains or losses of chromosomal material not found by metaphase or FISH analyses; and to correlate cytogenetics results with clinical, morphologic and immunophenotypic features of the disease, with cDNA microarray analysis of gene expression, and with outcome with new therapies. More than 275 patients have been entered on-study to date. Our initial analyses from 1997-2004 revealed clonal chromosome abnormalities in only 30-40 percent of cases using G-banded metaphase analysis, and demonstrated inferiority of e. coli lipopolysaccharide as a mitogen for the leukemic cells. Interphase FISH with a panel of five probes has confirmed or expanded the G-band findings in patients with abnormal clones, and has detected abnormalities in all but approximately 10% of patients tested to date. From 2002 through 2005 we performed CGH retrospectively on DNA isolated from patients previously entered on-study, and prospectively on DNA from new patients. CGH was less sensitive than interphase FISH in detecting submicroscopic deletions, but detected gains and losses of regions not probed by FISH. Combining G-banding, FISH, and CGH, we were able to find molecular cytogenetic abnormalities in more than 90% of B-CLL patients; FISH was clearly superior. Furthermore, dual hybridization with two probes and also sequential analyses in multiple patients have shown certain abnormalities present at diagnosis, and additional abnormalities at the time of transformation. Based upon these studies, we are now routinely performing interphase FISH on all patients. We are also quality-controlling new probes for addition to our routine FISH panel. Full G-banded karyotype analysis is done only if specifically indicated. Interphase FISH results are currently being used to classify patients with regard to risk, and in assignment to treatment protocols. Correlative studies of cytogenetics with clinical and other laboratory features at diagnosis and transformation, and with gene expression and response to treatment, are ongoing.