Discal cysts of the lumbar spine: a case report.

Discal cysts--intraspinal cysts communicating with an adjacent intervertebral disc--are an uncommon cause of lumbar radiculopathy. We report a case of discal cyst of the lumbar spine. The cyst contents were bloody and clotted rapidly; no disc materials were seen. Communication between the cyst and the intervertebral disc was detected. Histopathology of the cyst wall revealed fibrous connective tissue without synovial lining cells. We hypothesise that the discal cyst was formed by haemorrhage of the epidural venous plexus caused by separation of the peridural membrane by mechanical force transmitted by an annulus fibrosis fissure. The minute segmental motion caused by the affected disc may have stimulated continuous bleeding.

Solitary myofibroma of the lumbar vertebra: adult case.

We present the first known adult case of solitary myofibroma of bone, which affected a lumbar vertebra in a 33-year-old male. Radiography identified a purely lytic lesion with a sclerotic rim in the right pedicle of L1. CT showed an expansile lytic lesion with a sclerotic rim. MRI of the lesion revealed an isointense signal on T1-weighted images, an inhomogeneously hyperintense signal on T2-weighted images, and marked enhancement with gadolinium. Pathological study showed a mixed picture of nodular proliferation of spindle-shaped myoid cells and hemangiopericytomatous proliferation of short spindle/small round cells. The tumor cells were immunoreactive for smooth muscle actin and immunonegative for desmin. This case of solitary myofibroma of bone is exceptionally rare because of its occurrence in an adult older than 20 years of age and its location at an extra-craniofacial site.

Translin binds to the sequences adjacent to the breakpoints of the TLS and CHOP genes in liposarcomas with translocation t(12;6).

Myxoid and round-cell liposarcomas share the translocation t(12;16)(q13;p11) creating the TLS-CHOP fusion gene as a common genetic alteration. We previously reported several unique characteristics of genomic sequences around the breakpoints in the TLS and CHOP loci, and among them was the presence of consensus recognition motifs of Translin, a protein that associates with chromosomal translocations of lymphoid neoplasms. We further extended our search for Translin binding motifs in sequences adjacent to breakpoints and investigated whether Translin binds to these sequences in vitro by mobility-shift assay. Computer-assisted search found sequences highly homologous (>70%) with Translin binding motifs adjacent to the breakpoints in 10 out of 11 liposarcomas with the TLS-CHOP fusion genes. All of 13 oligonucleotides corresponding to the putative binding sequences in these cases bind to Hela cell extract and also recombinant Translin protein, although the binding affinity of each motif showed considerable differences. The DNA-protein complex formation was inhibited by non-labeled competitor or anti-Translin antibody, suggesting the specificity of the complex formation. Considering the high incidence and specific binding property, the presence of Translin binding motif may be one of the important determinants for the location of breakpoints in the TLS and CHOP genes in liposarcomas.

Characteristics of genomic breakpoints in TLS-CHOP translocations in liposarcomas suggest the involvement of Translin and topoisomerase II in the process of translocation.

Fusion of TLS/FUS and CHOP gene by reciprocal translocation t(12;16)(q32;q16) is a common genetic event found in myxoid and round-cell liposarcomas. Characterization of this genetic event was performed by three methods, Southern blot, RT-PCR, and genomic long-distance PCR in nine myxoid and three round-cell liposarcomas. All but one tumors showed genetic alternations indicating the fusion of TLS/FUS and CHOP gene. Two novel types of fusion transcripts were found, of which one lacked exon 2 sequence of CHOP gene, and the other lacked 3' half of exon 5 of TLS gene. The latter case was caused by a cryptic splicing site which was created by the genomic fusion. Detailed analyses genomic fusion points revealed several sequence characteristics surrounding the fusion points. Homology analyses of breakpoint sequences with known sequence motifs possibly involve in the process of translocation uncovered Translin binding sequences at both of TLS/ FUS and CHOP breakpoints in two cases. Translocations were always associated with other genetic alterations, such as deletions, duplications, or insertions. Short direct repeats were almost always found at both ends of deleted or duplicated fragments some of which had apparently been created by joining of sequences that flank the rearrangement. Finally, consensus topoisomerase II cleavage sites were found at breakpoints in all cases analysed, suggesting a role of this enzyme in creating staggered ends at the breakpoint. These data suggested that sequence characteristics may play an important role to recruit several factors such as Translin and topoisomerase II in the process of chromosomal translation in liposarcomas.

Amplification of the CDK4 gene in sarcomas: tumor specificity and relationship with the RB gene mutation.

Amplification of the CDK4 gene, which encodes a key molecule in the cell cycle, has been shown in some types of human neoplasms, including bone and soft tissue tumors. It is also reported that the CDK4 gene is coamplified with other sequences in the 12q13-15 region, including the MDM2 and SAS genes. Using 146 DNA samples derived from a variety of bone and soft tissue tumors, we have studied the pattern of amplification of these three genes, CDK4, MDM2, and SAS, to investigate whether there are any tumor type specific patterns of amplification. Amplification of at least one of these three genes was found in 18 tumors, and five different patterns of amplification were observed. Amplification of all of these three genes was detected in 9 cases. Amplification of the CDK4 gene without MDM2 amplification was observed in osteosarcomas and a chondrosarcoma but not in soft tissue tumors, whereas amplification of MDM2 gene alone was observed in malignant fibrous histiocytomas (MFHs), liposarcomas, and lipomas, but not in bone tumors. These results suggested that the CDK4 region is the primary target for amplification in bone tumors, whereas the MDM2 region is in soft tissue tumors. We also investigated the relationship of CDK4 amplification with retinoblastoma (RB) gene mutations in osteosarcomas, for which we have already performed the mutation analyses in detail. Interestingly, contrary to the prevailing theory that CDK4 amplification is an alternative mechanism for RB gene mutation, we found that three of four cases with amplification of the CDK4 gene showed loss of expression of the RB protein, one of which was proved to have an gross DNA alteration in the RB locus. This redundancy of mutations may indicate that the amplification of CDK4 may have some roles other than the inactivation of the RB protein in the development of osteosarcomas.

Establishment of an osteoblast-like cell line, MMC2, from p53-deficient mice.

An immortalized cell line exhibiting a well-differentiated osteoblast-like phenotype was established from calvaria of p53 tumor suppressor-deficient mice. This cell line, designated MMC2, showed several osteoblast-like properties such as high alkaline phosphatase activity, expression of type I collagen and osteocalcin mRNA, and differentiated in vitro to produce mineralized extracellular matrix. Alkaline phosphatase activity and the level of osteocalcin mRNA expression and the production of mineralized matrix were significantly enhanced by the addition of ascorbic acid. Although the cells proliferated rapidly and indefinitely, they did not grow in soft agar and were nontumorigenic in nude mice. These characteristics were equivalent to those observed in MC3T3-E1, a well-known osteoblast-like cell line. When inoculated in nude mice, however, MMC2 produced matured bone tissue, which was not observed in the case of MC3T3-E1. Expression of bone morphogenetic protein 2 and 4 and type IA receptor mRNA was demonstrated in cultured MMC2 cells. These results indicate that this new osteoblast-like cell line, MMC2, will be a unique material for the analysis of bone cell biology.

Fracture healing is a process independent of p53 function.

We studied the role of the tumor suppressor gene p53 in the process of fracture healing using mice with a p53 gene deficiency. Fractures produced in femoral shafts of mice without a functional p53 gene (p53-/-) healed as well as those in wildtype mice (p53 +/+), and no tumor development was observed at the fracture site even after complete bone union. Formation of granulation tissue and cartilage, ossification and remodeling into mature trabecular and cortical bone showed no abnormalities in p53-/- mice. Apoptotic cells were found to be sparse in the ossifying zone of the fracture callus using in situ DNA nick end-labeling in mice of both genotypes, without any significant difference. These results indicate that apoptosis in fracture healing, even if it does play a significant role, occurs through a p53-independent pathway.

Loss of heterozygosity and tumor suppressor gene mutations in chondrosarcomas.

Loss of heterozygosity (LOH) of chromosomes 13q and 17p and mutations of the retinoblastoma (Rb) and p53 gene were studied in 28 tumors from 22 patients with chondrosarcomas. Allele loss at polymorphic loci on 13q was found in 36% of tumors and the frequency was much higher in grade II (56%) or high grade (40%) tumors than in grade I tumors (18%). LOH of 17p, which was detected in 25% of all tumors, was of low frequency in low grade tumors (8% in grade I and 20% in grade II), whereas 80% of tumors with high grade tumors were positive for LOH. These observations may imply that LOH on 13q and 17p contributes to the tumor development at different stages. In low grade cases (grade I and II), tumor recurrence was observed more frequently in primary tumors with LOH on 13q (86%) than those without (8%), suggesting the significance of LOH analysis in the assessment of biological behavior of tumors. Structural alteration of the Rb gene was found in one dedifferentiated tumor, and point mutations of the p53 gene were found in all of five high grade tumors, indicating that high grade chondrosarcomas were genetically equivalent to other high grade sarcomas such as osteosarcomas.

MDM2 gene amplification in bone and soft-tissue tumors: association with tumor progression in differentiated adipose-tissue tumors.

We have studied 107 bone and soft-tissue sarcomas and 8 lipomas for amplification of the MDM2 gene. This gene was amplified in 3 out of 67 osteosarcomas, 3 out of 20 malignant fibrous histiocytomas, 4 out of 20 liposarcomas, and 4 out of 8 lipomas. The amplification was associated with overexpression of mRNA. In osteosarcomas, contrary to previous findings, all amplifications were observed in primary lesions. In liposarcomas, the amplification was seen exclusively in well-differentiated tumors with high frequency (4/5) but not in other subtypes (0/15). In addition, MDM2 amplification was also frequently found in deep-seated intra- or intermuscular lipomas (4/5). Hence, it is suggested that MDM2 amplification plays a significant role in the development of differentiated adipose-tissue tumors. Three well-differentiated liposarcomas with MDM2 amplification coexisted with high-grade dedifferentiated sarcomas, in which MDM2 amplification was also observed. Interestingly, in 2 of these cases, the grades of amplification correlated with the histological grades, indicating an important role of MDM2 overexpression in tumor progression.