Molecular Pathogenesis of Multiple Myeloma

我们的主要关注点是鉴定和描述多发性骨髓瘤(MM)肿瘤中IgH基因(染色体14q32)的易位。我们组装了一组36个EBV阴性的MM细胞系，发现：1)所有36个MM细胞系(HMCL)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断裂点的位置与大多数情况下B细胞特异性机制(主要是开关，但有时是体细胞过度突变)的错误一致；3)克隆的断裂点散布在大范围内，远离异常、过度表达的癌基因的1Mb；4)30个品系中至少有15个(50%)有两(10)个或三(5)个独立的IgH易位；5)4个染色体位点(位于11q13的细胞周期蛋白D1；位于6p21的细胞周期蛋白D3；位于4p16.3的FGFR3酪氨酸激酶受体和MM.SET；以及位于16q23的c-maf基本Zip转录因子)，每个易位约占MM的IgH易位的5-20%，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他人鉴定；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(60%为IgH，20%为2个独立的IGH，3个为独立的IGH，17%为Iglambda，无Igkappa，26%无Ig易位)。我们等人对MGUS癌前病变和恶性MM瘤的分析表明，47%的MGUS肿瘤和55~70%的MM肿瘤存在IgH易位，可能与疾病的分期有关。我们的工作假设是，在大约50%的肿瘤中，原发易位到Ig基因座提供了骨髓瘤分子发病机制中最初的永生化事件之一，并且发生在生发中心的浆细胞发育期间。此外，涉及其中一个Ig基因座的继发性易位--但缺乏由B细胞特异性重组机制介导的过程的特征--作为肿瘤进展事件发生。原发易位和继发易位似乎涉及不同的染色体配对(癌基因)，尽管可能有一些重叠。 第二个焦点是阐明我们的发现的意义，即在9例信息性HMCL中，尽管明显缺乏涉及c-myc基因的易位、重排或扩增，但存在L-myc或一个c-myc等位基因的选择性表达。结合FISH和SKY分析，我们有证据表明在我们所检查的28/32(88%)HMCL中存在L-myc(一个HMCL)或c-myc基因座的核型异常。由此可见，一种c-myc等位基因的选择性表达是肿瘤特有的复杂结构异常的结果(复杂的易位、插入、复制、倒位，经常累及3条不同的染色体，但并不总是一个免疫球蛋白基因座)，改变了两个L-myc或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HMCL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%)。部分原发肿瘤核型异常表现为c-myc异质性，1例肿瘤核型异常为N-myc。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。 第三个焦点是定义MM中的其他类型的遗传和表型异常。首先，我们已经证明在17/36(45%)HMCL中存在ras突变，这与原发MM肿瘤的发生率一致。其次，对于因t(4；14)易位而过度表达FGFR3的HMCL和原发MM肿瘤，我们发现大约10%的FGFR3突变和大约45%的ras突变，但没有一例同时存在这两种突变。与激活的ras类似，我们已经证明激活的FGFR3可以转化NIH3T3细胞。第三，我们已经在32%的HMCL和大约10%的非选择性肿瘤中发现了p18INK4c的双等位基因缺失，并提示这是MM肿瘤的一个子集的进展事件。最后，我们正在与Lou Staudt合作继续研究，使用淋巴芯片微阵列来确定我们的HMCL小组中的基因表达模式，目的是确定不同的易位是否会导致不同的基因表达模式和不同的肿瘤表型。

Our major focus has been to identify and characterize translocations to the IgH locus (chromosome 14q32) in multiple myeloma (MM) 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 (HMCL), 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 mostly but sometimes 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) four chromosomal loci (cyclin D1 at 11q13; cyclin D3 at 6p21; FGFR3 tyrosine kinase receptor and MM.SET at 4p16.3; and the c-maf basic zip transcription factor at 16q23) each account for about 5-20% 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 60%, 2 independent IgH in 20% and 3 independent IgH in none, Iglambda in 17%, Igkappa in none, and no Ig translocation in 26%). Analyses of premalignant MGUS and malignant MM tumor by ourselves and others show that IgH translocations are present in 47% of MGUS tumors and 55 to 70% of MM tumors, perhaps related to the stage of the disease. Our working hypothesis is that primary translocations to Ig loci provide one of the initial immortalizing events in the molecular pathogenesis of myeloma in about 50% of tumors, and occur during plasma cell development in germinal centers. In addition, secondary translocations involving one of the Ig loci - but lacking the hallmarks of a process mediated by a B cell specific recombination mechanism - occur as a tumor progression event. It appears that primary and secondary translocations involve different chromosomal partners (oncogenes), although there might be some overlap. 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 9 informative HMCL 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 L-myc (one HMCL) or c-myc locus in 28/32 (88%) HMCL that we have examined. Thus it seems clear that the selective expression of one c-myc allele 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 L-myc or 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 HMCL. 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, and one tumor had a karyotypic abnormality of N-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 shown that ras mutations are present in 17/36(45%) HMCL, consistent with the incidence in primary MM tumors. Second, for HMCL and primary MM tumors that overexpress FGFR3 due to the t(4;14) translocation, we find about 10% with mutations of FGFR3 and about 45% with mutations of ras, but none with mutations in both. Similar to activated ras, we have shown that activated FGFR3 can transform NIH3T3 cells. Third, we have identified bi-allelic deletion of p18INK4c in 32% of HMCL and about 10% of unselected tumors, and suggest that this is a progression event in a subset of MM tumors. Finally, we are continuing studies in collaboration with Lou Staudt to use the lymphochip microarray to determine the patterns of gene expression in our panel of HMCL, with a goal of determining whether different translocations result in different patterns of gene expression and different tumor phenotypes.