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

以前的G显带核型分析发现，在经过多克隆B细胞有丝分裂原适当的体外刺激后，研究的B-CLL患者中有40%-100%出现克隆性染色体异常。目前尚不清楚在不同系列中报告的异常频率的广泛差异是由于疾病特征的真正差异，还是仅仅是由于体外培养条件。常见的复发性染色体异常包括12三体、14q32重排、13q易位或缺失、6q缺失和11q缺失。最近的研究表明，间期荧光原位杂交(FISH)技术增加了对12三体、13q缺失、11q缺失和17p(P53)缺失的检测，表明这些变化确实发生在疾病的早期。关于细胞遗传学在B-CLL中的临床和预后意义的数据仍然有些有限，在某些情况下是相互矛盾的。然而，数据表明某些核型和/或FISH异常与B-CLL的特定临床病理亚型相关，并与疾病的病程有关。为了解决B-CLL核型改变的时间问题，以及这些改变可能的特异性和意义，我们正在对NCI转诊的散发性或家族性B-CLL患者进行前瞻性研究，以进行评估和可能的治疗。该项目的细胞遗传学特定目标是：确定从B-CLL患者的外周血中获取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G显带中期分析遗漏的克隆性异常细胞；确定比较基因组杂交(CGH)是否将检测中期或FISH分析未发现的染色体物质的得失；以及将细胞遗传学结果与疾病的临床、形态和免疫表型特征、基因表达的基因芯片分析以及新疗法的结果相关联。来自近200名参与研究的患者的数据显示，使用G显带中期酶分析的病例中，只有30%的患者出现克隆性染色体异常，并表明大肠杆菌脂多糖作为白血病细胞的有丝分裂原的能力较差。间期FISH和一组五个探针已经确认或扩大了克隆异常患者的G-带发现，并在迄今测试的所有患者中检测到异常，但约10%。从2002年到2005年，我们对先前进入研究的患者的DNA进行了回顾性的CGH，并对新患者的DNA进行了前瞻性的计算。在检测亚显微缺失方面，CGH不如间期FISH敏感，但可以检测到FISH未探测到的区域的得失。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。此外，两个探针的双重杂交和对多个患者的顺序分析显示，在诊断时存在某些异常，在转化时还有额外的异常。细胞遗传学的结果现在与诊断和转化时的临床和其他实验室特征以及基因表达和新治疗结果的基因微阵列分析相关联。该项目有可能获得有关临床细胞遗传学诊断技术的新信息，证明B-CLL患者的新的临床病理亚型，并确定对B-CLL的启动或进展至关重要的基因重排，该项目正在进行中。

Previous G-banded karyotype analyses have detected clonal chromosome abnormalities in 40-100 percent of B-CLL patients studied after appropriate in-vitro stimulation with polyclonal B-cell mitogens. It is currently unknown whether the wide variations in frequencies of abnormalities reported in different series are due to true differences in disease characteristics or merely to in-vitro culture conditions. Common recurring chromosomal abnormalities include 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 still somewhat limited, and in some cases conflicting. Nonetheless, 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 are conducting prospective studies of patients with sporadic or familial B-CLL referred to the NCI 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. Data from nearly 200 patients entered on-study reveal clonal chromosome abnormalities in only 30 percent of cases using G-banded metaphase analysis, and demonstrate 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 are able to find molecular cytogenetic abnormalities in more than 90% of B-CLL patients. 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. The cytogenetics results are now being correlated with clinical and other laboratory features at diagnosis and transformation, and with cDNA microarray analysis of gene expression and outcome with novel treatments. This project, which has the potential to yield new information regarding clinical cytogenetics diagnostic techniques, to demonstrate new clinicopathologic subsets of B-CLL patients, and to define genetic rearrangements critical to initiation or progression of B-CLL, is ongoing.