MOLECULAR PATHOGENESIS OF MULTIPLE MYELOMA

我们的主要焦点是鉴定和表征多发性骨髓瘤(MM)细胞系和肿瘤中IgH基因(染色体14q32.3)的易位。我们组装了36个EBV阴性的MM细胞系，发现：1)所有36个MM细胞系都存在Ig易位，包括IgH(33/36=92%)、Iglambda(5/23=23%)和Igkappa(0/21)；2)克隆的IgH断裂点的位置在大多数情况下与B细胞特异性机制(开关、VDJ重组、体细胞过度突变)的错误一致；3)克隆的断裂点散布在大范围内，距离异常、过度表达的癌基因有1Mb；4)至少有15个(50%)系有两(10)个或三(5)个独立的IgH易位；5)3个染色体位点(11q13处的细胞周期蛋白D1；4p16.3处的FGFR3酪氨酸激酶受体和MM.SET；以及16q23处的c-maf基本Zip转录因子)各占约10-20%的IgH易位，尽管4；14和14；16易位是常规核型检测不到的；6)至少有18(6)个其他易位对由我们和其他人发现；7)在一个由30个晚期肿瘤组成的小组中，易位的发生率较低(70%为IGH，20%为2个独立的IGH，无一例为3个独立的IGH，17%为Iglambda，无Igkappa，26%无易位)；8)我们有证据表明原发肿瘤中易位的异质性。我们的工作假设是，原发易位到Ig基因座经常--但不总是--在骨髓瘤的分子发病机制中提供了最初的永生事件之一，并且发生在生发中心的浆细胞发育期间。第二个焦点是阐明我们发现7个信息性MM细胞系中存在L-myc或一个c-myc等位基因选择性表达的意义(2例在相应的肿瘤中得到证实)，尽管明显缺乏涉及c-myc基因的易位、重排或扩增。从FISH和Sky分析的组合中，我们有证据表明在我们检查的23/28(82%)MM细胞系中有c-myc基因座的核型异常。因此，很明显，一种c-myc等位基因在MM细胞中的选择性表达是肿瘤特有的复杂结构异常(复杂的易位、插入、复制、倒位，经常涉及3条不同的染色体，但并不总是一个Ig基因座)的结果，它改变了两个亲本c-myc等位基因之一的染色体环境。在所有提供信息的病例中，很明显，myc结构异常存在于原发肿瘤和细胞系中。在晚期原发肿瘤样本中，c-myc异常的发生率似乎要低得多(45%)。一些原发肿瘤表现出c-myc核型异常的异质性。我们假设，复杂的核型异常似乎失调的c-myc很少-如果有的话-发生在肿瘤发生的早期事件。相反，c-myc的失调似乎是一个非常晚的进展事件，不是由B细胞特异的DNA修饰过程介导的。第三个重点是确定MM的其他类型的遗传和表型异常。首先，我们在一组36个MM系中筛选了ras和FGFR3突变，并在30个具有t(4；14)易位的原发MM肿瘤中筛选出6个FGFR3突变，初步结果与ras或FGFR3突变一致-但从未同时突变-在约40%的MM肿瘤中促进肿瘤进展。与激活的ras类似，我们已经证明激活的FGFR3可以转化NIH3T3细胞。其次，我们正在筛查细胞系中的p53突变。第三，与Lou Staudt一起，我们正在对我们30个特征良好的MM细胞系的mRNA表达进行淋巴芯片分析，并将这些结果与正常浆细胞以及MGUS和MM肿瘤进行比较。

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. We assembled a panel of 36 EBV negative MM cell lines, and find that: 1) Ig translocations are present in all 36 MM cell lines, including IgH (33/36 = 92%), Iglambda (5/23 = 23%), and Igkappa (0/21); 2) the location of cloned IgH breakpoints is consistent with errors of B cell specific mechanisms (switch, VDJ recombination, somatic hypermutation) in most cases; 3) cloned breakpoints are scattered over a large region, as far as 1 Mb from the dysregulated, overexpressed oncogene; 4) at least 15 of 30 (50%) lines have two (10) or three (5) independent IgH translocations; 5) 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 10-20% of IgH translocations in MM, even though the 4;14 and 14;16 translocations are not detected by conventional karyotypes; 6) there are a minimum of 18 (6 recurrent) other translocation partners identified by ourselves and others; 7) in a panel of 30 advanced tumors, translocations are somewhat less frequent (IgH in 70%, 2 independent IgH in 20% and 3 independent IgH in none, Iglambda in 17%, Igkappa in none, and no translocation in 26%); and 8) we have evidence of heterogeneity of translocations in primary tumors. Our working hypothesis is that primary translocations to Ig loci often - but not always - provide one of the initial immortalizing events in the molecular pathogenesis of myeloma, and occur during plasma cell development in germinal centers. In addition, secondary translocations involving one of the Ig loci occur as a late event, during tumor progression.A second focus is to clarify the significance of our finding that there is selective expression of L-myc or 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 23/28 (82%) 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, complex structural abnormality (complex translocation, insertion, duplication, inversion, with frequent involvement of 3 different chromosomes but not always an Ig locus) 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 incidence of c-myc abnormalities appears to be much lower (45%) in advanced, primary tumor samples. Some primary tumors show heterogeneity of the karyotypic abnormalities of c-myc. We have hypothesized that the complex karyotypic abnormalites that appear to dysregulate c-myc rarely - if ever- occur as an early event in tumorigenesis. Instead it appears that the dysregulation of c-myc occurs as a very late progression event that is not mediated by B cell specific DNA modification processes. 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 - but never both - contributing to tumor progression in about 40% of MM tumors. Similar to activated ras, we have shown that activated FGFR3 can transform NIH3T3 cells. Second, we are screening for p53 mutations in the cell lines. Third, with Lou Staudt, we are doing a lymphochip analysis of mRNA expression in 30 of our well-characterized MM cell lines, and comparing these results with normal plasma cells, and also MGUS and MM tumors.