Pleomorphic rhabdomyosarcoma (pRMS) is an aggressive histological variant of rhabdomyosarcoma, occurring predominantly in adults, usually older than 45 years of age, and represents only 2-5% of all adult soft tissue sarcomas. This tumor has a tendency to arise in large skeletal muscle of the extremities, mainly in the thigh. Like other pleomorphic sarcomas, pRMS generally has a poor prognosis because it has a highly aggressive nature and also a tendency to frequently metastasize to various sites, such as the brain and the lung [14
pRMS is the rarest of the three types of RMS, and only a few cytogenetic reports can be found in the literature [16
]. Though pRMS is not characterized by specific chromosomal translocations, unlike the other two variants (aRMS and eRMS). Instead, they are generally hyper-diploid with variable gains, rather than a specific translocation [18
]. However, some recurrent abnormalities have been reported in pRMS, including rearrangements involving chromosomes 1, 2, and 11 [19
]. HS-RMS-2 cells were hypertriploid and contained many numerical and structural abnormalities based on G-and Q-banding analysis. M-FISH readily depicted the precise chromosomes involved in various complex rearrangements, including 11 different unbalanced-translocated chromosomes, found in HS-RMS-2 cells. Many of the unbalanced translocations contained segments of chromosomes 1 and 8.
Tumor specific chromosomal abnormalities have been reported in a number of sarcomas, including t(11;22) in Ewing's sarcoma and peripheral primitive neuroectodermal tumor, t (12;16) in myxoid liposarcoma, and t(12;22) in clear cell sarcoma [17
]. These specific translocations are thought to be critically important in the pathogenesis of these tumors and result in the formation of fusion genes, such as EWS/ATF1
in clear cell sarcoma [17
]. In particular, the alveolar type of RMS frequently harbors a t(2;13), or t(1;13), thus resulting in the formation of a fusion gene, PAX3-FKHR
]. On the other hand, eRMS exhibit loss of heterozygosity at 11p15.5, and the loss of a putative tumor suppressor gene located at this location has been proposed to have a causal role in eRMS [23
]. Interestingly, loss of this region was also observed in the HS-RMS-2 cell line based on the CGH analysis. Not only pRMS but also other pleomorphic sarcomas usually exhibit complex karyotypes with no specific pattern [14
]. Further studies and the accumulation of similar cases will be required to determine whether there are specific translocations or genes pathognomonic for pRMS.
The CGH study showed numerous DNA gains and losses in this cell line. Gains of chromosomes 1, 8 and 18, and losses of chromosomes 2, 5, 11, 13, 14, 18 and 19 were consistent with the data of Li et al. [18
]. Remarkably, eight high-level amplifications were detected at three regions on chromosomes 1, two regions on chromosome 8, and one region each on chromosomes 11, 12 and 18. Generally, oncogene amplification is a major genomic force contributing to the development of human cancers, including RMS [17
]. Amplified oncogenes located at regions of high-level copy number gains may be related to pathogenesis of this tumor. The co-existence of high-level gains of 8 different chromosomal regions in one tumor cell is notable. These high-level gains are associated with the amplification of multiple oncogenes, including JUN
(11q13.3) and MDM2
(12q14.3-q15), each of which is commonly amplified in various types of solid tumors [17
]. The amplified oncogenes observed in the HS-RMS-2 cell line may contribute to the pathogenesis of pRMS and will be explored in future work. The co-amplification of multiple oncogenes, and the wide distribution of the amplified genes within the genome of the HS-RMS-2 cells suggest that multiple oncogenes may cooperate oncogenically in these cells.
The identification of amplified sequences of DNA using CMD-generated probe is a useful approach for detecting amplified oncogenes [9
]. The CMD probe used here was generated from one acro-centric long marker chromosome from a HS-RMS-2 cell. The CMD-generated probe hybridized to large segments in several marker chromosomes containing putative hsrs, while the hybridization to metaphase spreads from lymphocytes provided information regarding the native location of the DNA present in the hsr. The oncogenes that contributed the hsr formation were revealed by dual-color FISH using cosmid and BAC probes. FISH using the CMD probe suggested that there are at least three oncogenes located in the hsr. In addition, FISH experiments using commercially available probes, demonstrated that six oncogenes tested (JUN, MYC, INT2, CCND1, MDM2
, are amplified in HS-RMS-2 cells. JUN
have been implicated in the growth of RMS [33
]. Inhibition of MYC
decreases RMS tumorigenicity and rescues myogenic differentiation [35
is involved in several biological processes such as cell proliferation, differentiation, and apoptosis, and MYC
deregulation is observed in a large number of human tumors [36
encodes a protein belonging to the fibroblast growth factor family, which is thought to be involved in the pathogenesis of some sarcomas [17
]. In addition, amplification of 12q13-q14 has been reported in aRMS and has been associated with local tumor invasion [26
]. Taken together, these findings suggest that the highly amplified genes detected in our cell line may have some related function, such as perturbing signal transduction related to cell growth and differentiation. Tumor subtypes may also be distinguished by their propensity for amplifying oncogenes, thus suggesting that the particular types of genomic instability present in a tumor are important determinants of how expression of an oncogene might be altered. Moreover, tumors with gene amplifications are often associated with poor prognosis [26
]. Co-amplification of INT2
, each located in 11q13, are frequently observed in sarcomas. MALT
gene amplification, on the other hand, has been reported MALT lymphoma [37
]. Amplification of MDM2
is important because it results in degradation of p53 [38
]. Oncogenes located at the three remaining amplified regions revealed by CGH, i.e., 1p36.1-p36.2, 1q21-q31, and 8q13-q21, remain to be elucidated.
The previously established HS-RMS-1 cell line did not appear to have any hsrs, and CGH did not detect any gene amplification. According to our clinical reports of HS-RMS-1 and HS-RMS-2 derived patients, the former patient (HS-RMS-1) deceased at about 2.5 years after surgery, however, latter (HS-RMS-2) died at about 6 months. This implies that the tumor from which the HS-RMS-2 line with several hsrs was derived from a more aggressive or more undifferentiated tumor than the one that gave rise to the HS-RMS-1 cell line. Further study of the HS-RMS-2 cell line may provide insights into the mechanisms leading to genomic instability, hsr formation, and co-amplification of oncogenes in relation to pRMS.