MOLECULAR PATHOGENESIS OF MULTIPLE MYELOMA

他说：“我们的主要工作重点是找出和 描述IgH基因座(染色体14q32.3)易位的特征 在多发性骨髓瘤(MM)细胞系和肿瘤中。直到最近， IgH易位仅在常规方法中很少被发现 多发性骨髓瘤及其配对染色体的核型分析 极少鉴定(14Q+)。确定免疫球蛋白的频率 易位和配对染色体的身份(和 癌基因)参与MM的发病机制，我们组装了一个 30株EBV阴性多发性骨髓瘤细胞系。对这些线进行了分析 通过结合常规核型，FISH使用IgH和 配对染色体探针，光谱核型分析(SKY)，以及 Southern印迹结合克隆鉴定分子易位 断点。虽然这些研究尚未完全完成，但我们 可以得出这样的结论：1)每条线有一个或多个IGH 易位；2)大多数IGH易位涉及IGH开关 区域；3)克隆断点散布在大区域， 往往从数百kb的失控、过度表达 癌基因；4)至少7个品系有两个独立的免疫球蛋白 易位；5)我们已经报告了第一例 同时失调两种潜能的易位 癌基因(t[4；14]易位，存在调控失调 FGFR3和一个名为MM.SET的新基因，它与 MLL基因)；6)相同的分子易位 我们能够获得的4个系的原代肿瘤细胞 肿瘤；7)三个染色体位点(11q13处的细胞周期蛋白D1；FGFR3 酪氨酸激酶受体和MM.SET位于4p16.3；c-maf 基本的ZIP转录因子在16q23)各约占20% MM中的IgH易位，即使4；14和14；16 传统的核型检测不到易位；8)我们的 结果，与其他实验室的结果一起证明 另外6个重复的染色体配对和 9例MM中涉及Ig易位的单发伴侣 最近我们开发了鱼类探测器，这将使我们能够 高效检测IgKappa和IgLambda易位。我们是 继续使用IGL和IGL进行其他Sky和FISH分析 探针，以及它们的分子克隆和表达研究 线条和原发肿瘤样本以获得更完整的洞察 Ig易位在多发性骨髓瘤中的作用我们的工作假设是 易位到Ig基因座提供了最初永垂不朽的 骨髓瘤分子发病机制中的事件，并发生在 生发中心的浆细胞发育。 第二个重点是澄清我们的发现的意义。 有一个c-myc等位基因在7 信息性多发性骨髓瘤细胞系(在 2例)尽管表面上没有易位， 涉及c-myc基因的重排或扩增。从一个 结合鱼类和天空分析，我们有证据表明 几乎所有MM细胞中c-myc基因座的核型异常 我们已经检查过的线条。因此，似乎很明显，选择性的 一个c-myc等位基因在MM系中的表达是一种 改变染色体的肿瘤特异性结构异常 两个亲本c-myc等位基因之一的上下文。总而言之，信息丰富 4例中，很明显，myc结构异常出现在 原发肿瘤以及细胞系中的肿瘤。分子 C-myc结构蛋白的发生机制和发生时间 多发性骨髓瘤发病过程中的异常情况尚待确定。 第三个关注点是定义其他类型的基因和 MM的表型异常。首先，我们对ras进行了筛查。 36个MM系的FGFR3突变，以及FGFR3的突变 30例具有t(4；14)基因的原发多发性骨髓瘤中的6例突变 易位，初步结果与突变相一致 RAS或FGFR3在约40%的肿瘤进展中起作用 MM肿瘤。其次，我们正在筛查P53和Bax突变 在细胞系中。第三，与Lou Staudt一起，我们正在做淋巴芯片 28例恶性多发性骨髓瘤的基因表达分析 细胞系。“

"Our major focus has been to identify and characterize translocations to the IgH locus (chromosome 14q32.3) in multiple myeloma (MM) cell lines and tumors. Until recently, IgH translocations were detected only infrequently by conventional karyotypic analysis of MM tumors, and the partner chromosome was identified rarely (14q+). To determine the frequency of IgH translocations and the identity of partner chromosomes (and oncogenes) involved in the pathogenesis of MM, we assembled a panel of 30 EBV negative MM cell lines. These lines were analyzed by a combination of conventional karyotypes, FISH using IgH and partner chromosome probes, spectral karyotyping (SKY), and Southern blotting plus cloning to identify molecular translocation breakpoints. Although these studies are not yet fully completed, we are able to conclude that: 1) each line has one or more IgH translocations; 2) most IgH translocations involve IgH switch regions; 3) cloned breakpoints are scattered over a large region, often hundreds of kb from the dysregulated, overexpressed oncogene; 4) at least 7 lines have two independent IgH translocations; 5) we have reported the first example of a translocation that simultaneously dysregulates two potential oncogenes (with t[4;14] translocations, there is dysregulation of FGFR3 and a novel gene called MM.SET that is related to the MLL gene); 6) the same molecular translocation was present in the primary tumor cells of the 4 lines for which we were able to obtain tumor; 7) three chromosomal loci (cyclin D1 at 11q13; FGFR3 tyrosine kinase receptor and MM.SET at 4p16.3; and the c-maf basic zip transcription factor at 16q23) each account for about 20% of IgH translocations in MM, even though the 4;14 and 14;16 translocations are not detected by conventional karyotypes; 8) our results, together with results from other laboratories demonstrate the involvement of 6 additional recurrent chromosomal partners and 9 single incidence partners involved in an Ig translocation in MM. Recently we have developed FISH probes that will enable us to efficiently detect IgKappa and IgLambda translocations. We are continuing additional SKY and FISH analyses with IgH and IgL probes, as well as molecular cloning and expression studies on these lines and primary tumor samples to gain a more complete insight into the role of Ig translocations in MM. Our working hypothesis is that translocations to Ig loci provide one of the initial immortalizing events in the molecular pathogenesis of myeloma, and occur during plasma cell development in germinal centers." "A second focus is to clarify the significance of our finding that there is selective expression of one c-myc allele in 7 informative MM cell lines (confirmed in the corresponding tumor in 2 cases) despite the apparent absence of a translocation, rearrangement, or amplification involving the c-myc locus. From a combination of FISH and SKY analyses, we have evidence for karyotypic abnormalities of the c-myc locus in virtually all MM cell lines that we have examined. Thus it seems clear that the selective expression of one c-myc allele in MM lines is a consequence of a tumor specific structural abnormality that alters the chromosomal context of one of the two parental c-myc alleles. In all informative cases, it is clear that the myc structural abnormality was present in the primary tumor as well as in the cell line. The molecular mechanism and time of occurrence of c-myc structural abnormalities during pathogenesis of MM remain to be determined" "A third focus is to define other kinds of genetic and phenotypic abnormalities in MM. First, we have screened for ras and FGFR3 mutations in a panel of 36 MM lines, and for FGFR3 mutations in 6 of 30 primary MM tumors that have the t(4;14) translocation, with preliminary results consistent with mutation in ras or FGFR3 contributing to tumor progression in about 40% of MM tumors. Second, we are screening for p53 and bax mutations in the cell lines. Third, with Lou Staudt, we are doing a lymphochip analysis of mRNA expression in 28 of our well-characterized MM cell lines."