Molecular Pathogenesis of Multiple Myeloma

我们的主要重点是识别和表征多发性骨髓瘤（MM）肿瘤中IgH基因座（染色体14 q32）的易位。我们组装了一组36个EBV阴性的MM细胞系，发现：1）IG易位存在于所有36个MM细胞系（HMCL）中，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B细胞特异性机制的错误一致3）克隆的断裂点分散在一个大的区域，离失调的、过表达的癌基因远达1 Mb; 4）30个品系中的至少15个（50%）具有两个（10）或三个（5）独立的IgH易位;（11 q13的cyclin D1，6p 21的cyclin D3，4p16.3的FGFR 3酪氨酸激酶受体和MM.SET;和16 q23处的c-maf碱性zip转录因子）各自占MM中IgH易位的约5-20%，尽管常规核型未检测到4;14和14;16易位;（6）至少有18个（6）反复出现的由自己和他人确定的其他易位伴侣; 7）在一组30例晚期肿瘤中，易位的发生率稍低IgH阳性率为60%，独立IgH阳性率为20%，独立IgH阴性率为3%，Iglambda阳性率为17%，Igkappa阳性率为0%，无IG易位发生率为26%。我们对MGUS癌前病变和恶性MM肿瘤的分析表明，MGUS肿瘤中47%和MM肿瘤中55 ~ 70%存在IgH易位，这可能与疾病的分期有关。我们的工作假设是，原发性易位到IG基因座提供了一个最初的永生化的事件在骨髓瘤的分子发病机制在约50%的肿瘤，并发生在浆细胞发育在生殖中心。此外，涉及IG基因座之一的继发性易位-但缺乏由B细胞特异性重组机制介导的过程的标志-作为肿瘤进展事件发生。原发性和继发性易位涉及不同的染色体伴侣（癌基因），尽管可能有一些重叠。 第二个重点是澄清我们的发现，有选择性表达的L-myc或一个c-myc等位基因在9信息HMCL的意义，尽管明显缺乏易位，重排，或扩增涉及c-myc基因座。通过FISH和SKY分析的结合，我们有证据表明，我们检查的28/32（88%）HMCL中L-myc（一种HMCL）或c-myc基因座存在核型异常。因此，似乎清楚的是，一个c-myc等位基因的选择性表达是肿瘤特异性的复杂结构异常（复杂易位、插入、复制、倒位，经常涉及3个不同的染色体，但不总是IG基因座）的结果，其改变了两个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。c-myc异常的发生率在晚期原发性肿瘤样本中似乎要低得多（45%）。一些原发性肿瘤显示c-myc核型异常的异质性，并且一个肿瘤具有N-myc核型异常。我们假设，复杂的核型异常，似乎失调c-myc很少-如果有的话-发生在肿瘤发生的早期事件。相反，似乎c-myc的失调是作为一种非常晚期的进展事件发生的，它不是由B细胞特异性DNA修饰过程介导的。 第三个重点是确定其他类型的遗传和表型异常的MM。首先，我们已经表明，ras突变存在于17/36（45%）HMCL，与原发性MM肿瘤的发病率一致。第二，对于由于t（4;14）易位而过度表达FGFR 3的HMCL和原发性MM肿瘤，我们发现约10%具有FGFR 3突变，约45%具有ras突变，但两者均无突变。与活化的ras类似，我们已经表明活化的FGFR 3可以转化NIH 3 T3细胞。第三，我们在32%的HMCL和约10%的AML肿瘤中鉴定了p18 INK 4c的双等位基因缺失，并表明这是MM肿瘤亚组的进展事件。最后，我们与Lou施陶德合作继续研究，使用淋巴芯片微阵列来确定我们的HMCL组中的基因表达模式，目的是确定不同的易位是否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.