Recurrent chromosomal imbalances and structurally abnormal breakpoints within complex karyotypes of malignant peripheral nerve sheath tumour and malignant triton tumour: a cytogenetic and molecular cytogenetic study.

BACKGROUND: Cytogenetic studies of malignant peripheral nerve sheath tumours (MPNSTs) and malignant triton tumours (MTTs) are rare. AIMS: To undertake cytogenetic analysis of these tumours. METHODS: Conventional cytogenetic analysis of 21 MPNSTs and MTTs from 17 patients (nine with peripheral neurofibromatosis (NF1)) was carried out using standard culture and harvesting procedures. For a more precise identification of composite structural rearrangements and marker chromosomes, spectral karyotypic analysis (SKY) was applied to a subset of cases. In addition, EGFR gene copy number was assessed by fluorescence in situ hybridisation (FISH) analysis in a subset of cases. RESULTS: Cytogenetic analysis revealed predominantly complex karyotypes. SKY analysis was useful in further defining many structural anomalies. Structural aberrations most frequently involved chromosomal bands or regions 1p31-36, 4q28-35, 7p22, 11q22-23, 19q13, 20q13, and 22q11-13. Overall, loss of chromosomal material was much more common than gain. Loss of chromosomes or chromosomal regions 1p36 (48%), 3p21-pter (52%), 9p23-pter (57%), 10 (48%), 11q23-qter (48%), 16/16q24 (62%), 17(43%), and 22/22q (48%), and gains of 7/7q (29%) and 8/8q (29%) were most prominent. These gains and losses were distributed equally between MPNST and MTT, demonstrating that these entities are similar with respect to recurrent genomic imbalances. Similarly, none of the recurrent chromosomal breakpoints or imbalances was restricted to either NF1 associated or sporadic MPNSTs. FISH analysis was negative for amplification. CONCLUSIONS: These cytogenetic and molecular cytogenetic findings expand the knowledge of chromosomal alterations in MPNST and MTT, and point to possible recurring regions of interest.

Prognostic impact of P53 status, TLS-CHOP fusion transcript structure, and histological grade in myxoid liposarcoma: a molecular and clinicopathologic study of 82 cases.

PURPOSE: A specific TLS-CHOP fusion gene resulting from the t(12;16) is present in at least 95% of myxoid liposarcomas (MLS). Three common forms of the TLS-CHOP fusion have been described, differing by the presence or absence of TLS exons 6-8 in the fusion product. Type 5-2 (also known as type II) consists of TLS exons 1-5 fused to CHOP exon 2; type 7-2 (also known as type I) also includes TLS exons 6 and 7 in the fusion, whereas type 8-2 (also known as type III) fuses TLS exons 1-8 to CHOP exon 2. We sought to determine the impact of TLS-CHOP fusion transcript structure on clinical outcome in a group of well-characterized MLS cases. We also analyzed P53 status, because this parameter has been found to have a significant prognostic impact in other sarcomas with chromosomal translocations. METHODS: We analyzed TLS-CHOP fusion transcripts by reverse-transcription PCR using RNA extracted from frozen tissue in 82 MLS confirmed previously to harbor a CHOP rearrangement either by Southern blotting or by cytogenetic detection of the t(12;16). Parameters analyzed included age, location, size, percentage of round cell (RC) component, areas of increased cellularity, necrosis, and surgical margins. In 71 (87%) cases, adequate tumor tissue was available for immunohistochemical analysis of P53 status, using DO7 antibody. The Kaplan-Meier method, log-rank, and Cox regression tests were used for survival analyses. RESULTS: Most MLS were >10 cm (73%), arising in the thigh (70%), and localized at presentation (89%). RC component was <5% in 47 (57%) cases and > or =5% in 35 (43%). The TLS-CHOP fusion transcript was type 5-2 in 55 (67%), type 7-2 in 16 cases (20%), and type 8-2 in 8 (10%). One tumor had a unique variant fusion, between exon 6 TLS and exon 2 CHOP. Two other cases (2%) showed an EWS-CHOP fusion transcript. Overexpression of P53 (defined as > or =10% nuclear staining) was detected in 12 (17%) cases. High histological grade (defined as > or =5% RC; P < 0.01), presence of necrosis (> or =5% of tumor mass; P < 0.05), and overexpression of P53 (P < 0.001) correlated with reduced metastatic disease-free survival in localized tumors. The presence of negative surgical margins (P < 0.01) and extremity location (P = 0.02) were found to be significant in predicting local recurrence in the entire group as well as localized cases by univariate and multivariate analysis. Although there was no significant correlation between TLS-CHOP transcript type and histological grade or disease-specific survival, an association was found between the P53 status and type 5-2 fusion (P < 0.01). CONCLUSION: In contrast to some other translocation-associated sarcomas, the molecular variability of TLS-CHOP fusion transcript structure does not appear to have a significant impact on clinical outcome in MLS. Instead, high histological grade (> or =5% RC), presence of necrosis, and P53 overexpression are predictors of unfavorable outcome in localized MLS.

Translocation t(1;3)(p36.3;q25) is a nonrandom aberration in epithelioid hemangioendothelioma.

The cytogenetic findings for two epithelioid hemangioendotheliomas are reported. An identical chromosomal translocation involving chromosomes 1 and 3 [t(1;3)(p36.3;q25)] was detected in both cases of epithelioid hemangioendothelioma, possibly representing a characteristic rearrangement for this histopathologic entity. The presence of clonal karyotypic abnormalities supports a neoplastic origin for the epithelioid variant of hemangioendothelioma. Identification of the 1;3 translocation may be useful diagnostically. Should additional studies confirm these data, this could lead to the identification of the gene(s) central to this neoplastic process.

Cytogenetic instability, predominantly involving chromosome 1, is characteristic of elastofibroma.

Elastofibroma, an unusual pseudotumor composed of excessive collagen and abnormal elastic fibers, has rarely been subjected to cytogenetic analysis. Only two cases have been previously defined, both of which demonstrated nonclonal abnormalities. In the present study, three cases of elastofibroma were cytogenetically analyzed. Abnormalities of the short arm of chromosome 1 were seen in all three cases (either clonally or as the most frequently involved region among nonclonal aberrations). In addition, a translocation involving chromosomes 8 and 12 was detected as a clonal rearrangement in one of the three cases. The observation of clonal abnormalities in elastofibroma suggests that this lesion may represent a neoplastic rather than a reactive process.

Extra copies of chromosomes 7, 8, 12, 19, and 21 are recurrent in adamantinoma.

Adamantinoma of long bones is a rare neoplasm predominantly involving the tibia. Cytogenetic studies of adamantinoma are few. Cytogenetic or molecular cytogenetic analysis of four adamantinomas, and a review of eleven cases in the literature reveals extra copies of chromosomes 7, 8, 12, 19, and 21 as recurrent in this neoplasm. Adamantinoma may be confused with a variety of primary and metastatic epithelial and mesenchymal neoplasms. Observation of these aneuploidies may be useful in establishing the diagnosis of adamantinoma.

Cytogenetic findings in a case of epithelioid sarcoma and a review of the literature.

Cytogenetic studies of epithelioid sarcoma, a rare malignant soft tissue neoplasm of adolescents and young adults, are few. A characteristic anomaly has not yet been identified for this sarcoma. In this study, cytogenetic studies of a primary epithelioid sarcoma of a 15-year-old male revealed the following abnormalities: t(6;8)(p25;q11.2) and add(7)(p15).

Recurrent anomalies of 6q25 in chondromyxoid fibroma.

Chondromyxoid fibroma is a rare benign bone tumor most commonly arising in the metaphysis of long bones in young adults. Histopathologically, chondromyxoid fibroma may be difficult to distinguish from other cartilaginous neoplasms. Recently, a pericentric inversion of chromosome 6 [inv(6)(p25q13)] has been proposed as a specific genetic marker for chondromyxoid fibroma. In this study, cytogenetic and spectral karyotypic analyses of 2 chondromyxoid fibroma cases showed clonal abnormalities of chromosome 6 but at a breakpoint on the long arm (q25) distal to that described in the pericentric inversion. These findings suggest that several distinct breakpoints on chromosome 6 are nonrandomly involved in chondromyxoid fibroma.

Interleukin-1beta upregulates MMP-9 expression in stromal cells of human giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1beta (IL-1beta) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1beta and MMP-9. The addition of monoclonal antibody (mAb) against IL-1beta partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescence showed that GCT stromal cells did not express MMP-9, although treatment with IL-1beta induced MMP-9 expression in a dose-dependent manner, and the secretion peaked 24 h after stimulation and then plateaued. Studies with cycloheximide, a protein synthesis inhibitor, demonstrated that de novo protein synthesis is required for IL-1beta induced MMP-9 expression. Moreover, nuclear run-on analysis has revealed that IL-1beta significantly increased MMP-9 gene transcription in GCT stromal cells. The data suggest that IL-1beta secreted by the multinucleated giant cells in GCT may be one of the factors responsible for the induction of MMP-9 at the transcriptional level in GCT stromal cells in vivo. We conclude that GCT has a self-stimulatory system for the production of MMP-9, and the ability of stromal cells to produce MMP-9 with appropriate stimuli, such as IL-1beta, and possibly in concert with other cytokines may contribute to the aggressive and potentially malignant behavior of GCT.

The cytotoxic effects of single-stranded telomere mimics on OMA-BL1 cells.

Telomerase is a ribonucleoprotein that adds 5'-d(TTAGGG)-3' hexameric repeats onto the 3' ends of chromosomes. High telomerase activity has been associated with immortal cells, transformed cells, mitogenic stimulation, and proliferative diseases. It is not clear what phenotype would be observed by transient inhibition of telomerase. Studies were designed to inhibit telomerase activity using a series of S-ODN telomere sequence motifs. The studies evaluated the length, hydrogen bonding, and sequence requirements of telomerase inhibition using the TRAP assay and a bioassay measuring cell viability following exposure to the compounds. In addition, we have also studied the role of the 3' end and secondary structure of telomere mimics on telomerase inhibition. Observations reveal that sensitivity to the S-ODNs may not require hybridization to an antisense target but required guanine nucleotides on the 3' end for cells in culture and telomerase inhibition in vitro. The importance of H bonding and the requirement for a free 3' end for the activity of these compounds has also been demonstrated. However, transient inhibition of telomerase is not cytotoxic to all immortal cells and is not sufficient to explain the mechanism of cytotoxicity of these short oligonucleotides.

Trisomies 8 and 20 characterize a subgroup of benign fibrous lesions arising in both soft tissue and bone.

Trisomy 8 and trisomy 20 are nonrandom aberrations in desmoid tumors. The presence of these trisomies in related benign fibrous lesions of bone has not been previously addressed. In this study, 22 specimens from 19 patients diagnosed with desmoid tumor, desmoplastic fibroma, periosteal desmoid tumor, osteofibrous dysplasia, or fibrous dysplasia were examined by cytogenetic analysis of short-term cultures and bi-color fluorescence in situ hybridization of cytological touch preparations or paraffin-embedded tissue with centromeric probes for chromosomes 8 and 20. Trisomy 8 and trisomy 20 were detected by molecular cytogenetic methodologies in 15 specimens, including 10 primary bone lesions. Traditional cytogenetic analysis revealed trisomy 8 in two cases of osteofibrous dysplasia. Our findings demonstrate that trisomy 8 and trisomy 20 are also nonrandom aberrations in histologically similar, but clinically distinct, benign fibrous lesions of bone.

Adamantinoma-like Ewing's sarcoma: genomic confirmation, phenotypic drift.

Ewing's sarcoma, a highly malignant neoplasm, is characterized by an 11;22 translocation [t(11;22) (q24;q12)], resulting in the fusion of genes FLII and EWS. Adamantinoma of extragnathic bones, a low-grade malignant neoplasm with epithelial features, is not typically considered in the differential diagnosis of Ewing's sarcoma. In this study, three osseous Ewing's sarcomas with histological, immunohistochemical, or ultrastructural epithelial features were subjected to reverse transcription-polymerase chain reaction and sequencing studies for the Ewing's sarcoma molecular rearrangement. (Two of the three cases were originally described as adamantinomas or nontypical Ewing's sarcoma before the availability of genetic characterization.) In addition, traditional cytogenetic analysis and a unique combined interphase molecular cytogenetic/ immunocytochemical approach with bicolor 11;22 translocation breakpoint flanking probes (cosmids) and pancytokeratin antibodies were performed on one neoplasm. At(11;22) (q24;q12) was found in one neoplasm and a type II EWS/FLI-1 fusion transcript was detected in all three neoplasms. The combined genetic/immunocytochemical approach revealed the presence of the 11 ;22 translocation in the nuclei of cytokeratin immunoreactive cells. These genotypic and phenotypic findings delineate a novel Ewing's sarcoma histologic variant, "adamantinoma-like Ewing's sarcoma."

Transcriptional regulation of MMP-9 expression in stromal cells of human giant cell tumor of bone by tumor necrosis factor-alpha.

We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.

Involvement of 3q21 in nodular fasciitis.

Cytogenetic data on nodular fasciitis are sparse. We present a case of this lesion and discuss our results in view of previously reported findings in nodular fasciitis and other benign mesenchymal lesions.

Clear cell sarcoma or malignant melanoma of soft parts: molecular analysis of microsatellite instability with clinical correlation.

Malignant melanoma of soft parts, also termed clear cell sarcoma (CCS), is a rare malignancy of neural crest origin which is different from cutaneous malignant melanoma. Although a translocation involving chromosomes 12 and 22 is characteristic of clear cell sarcoma and not malignant melanoma, there are a paucity of methods to differentiate the two. Therefore, a study of microsatellite instability (MIN) was undertaken to determine if mechanisms of DNA mismatch repair can differentiate these malignancies. MIN has been described in a variety of malignancies including 25% of malignant melanomas. Paraffin-embedded neoplastic and non-neoplastic cells were obtained from 11 individuals (five males; six females; age range from seven to 60 years) with CCS. Isolated DNA was PCR amplified at 17 separate microsatellite loci using radioactive-labeled primers. Tumor tissue was compared to normal tissue for each analysis. No MIN was detected. Loss of heterozygosity was detected in only one patient at a single locus (IFNA). The lack of MIN in clear cell sarcoma further defines the distinction between this tumor and malignant melanoma. Clinically, local recurrence and metastasis were indicators of poor outcome. The size of the tumor was not a significant prognostic indicator. Local recurrence, satellitosis, or nodal metastasis was not proven to be uniformly fatal. Utilization of chemotherapy and/or radiation demonstrated no obvious survival advantage. The histologic parameters of mitotic rate and the presence of necrosis were not prognostic. Limb-preserving surgical procedures were as effective as amputation for local disease control. The actuarial survival rate was calculated to be 48% at five years.

Comparison of the Nebraska collar, a new prototype cervical immobilization collar, with three standard models.

OBJECTIVES: To determine whether a novel immobilization collar called the Nebraska collar would restrict motion of the cervical spine better than three traditional designs: the Philadelphia collar, the sterno occipital mandibular immobilizer (S.O.M.I.), and the Lehrman-Minerva cervical orthosis. DESIGN: Cervical spine radiographs and a compass were used to assess motion allowed by four separate cervical collars placed on volunteers. SETTING: University-affiliated level one trauma center. PATIENTS/PARTICIPANTS: Fourteen paid volunteers (six females and eight males) between the ages of twenty and thirty-five years (mean twenty-five years) were recruited. MAIN OUTCOME MEASUREMENTS: The maximum amount of flexion, extension, and lateral bending permitted by each collar was assessed by cervical radiographs taken of the volunteers while wearing each of the four collars. Maximum rotation was measured with a compass positioned on the top of the head of the volunteers and oriented in the horizontal plane. RESULTS: The Nebraska collar restricted rotation (p < 0.0001) and lateral bending (p < 0.0001) significantly more than did the other three orthoses. In total maximum extension from occiput to C7, the Nebraska collar was found to be more restrictive than the Philadelphia collar (p < 0.05) and the S.O.M.I. (p < 0.05). In total maximum flexion, there was no statistically significant difference among the four collars. When the total maximum flexion-to-extension motion was measured, both the Nebraska and Lehrman-Minerva cervical collars were found to be more restrictive than the Philadelphia collar (p < 0.05). CONCLUSION: The new Nebraska collar provides stabilization that is significantly more rigid than the other models tested, with no difference in patient comfort.

Significance of abnormalities of chromosomes 5 and 8 in chondroblastoma.

Tumor specific chromosomal abnormalities have been identified in several histologic subtypes of benign and malignant bone tumors. These anomalies have proven to be useful diagnostically. Characterization of recurrent chromosomal abnormalities also has provided direction for molecular investigations of pathogenetically important genes. Cytogenetic reports of chondroblastoma, a rare benign bone tumor, are few. Cytogenetic analysis of a benign and a malignant chondroblastoma in this study revealed the following abnormal chromosomal complements: 47,XY,+5,t(5;5)(p10;q10) and 45, XY,del(2)(p23),del(3)(q23q27),dup(8)(q12q21.), del(11) (q14q23), -13, add (17) (q25) x 2, respectively. Although a specific chromosomal abnormality has not yet emerged for chondroblastoma, abnormalities of chromosomes 5 and 8 have been reported previously in this neoplasm, suggesting preferential involvement of these two chromosomes.

Clonal karyotypic abnormalities of the hereditary multiple exostoses chromosomal loci 8q24.1 (EXT1) and 11p11-12 (EXT2) in patients with sporadic and hereditary osteochondromas.

BACKGROUND: Osteochondroma most frequently arises sporadically and as a solitary lesion, but also may arise as multiple lesions characterizing the autosomal dominant disorder hereditary multiple exostoses (HME) and the contiguous gene syndromes Langer-Giedion and DEFECT-11 syndromes. HME is genetically heterogeneous with association of three loci including 8q24.1 (EXT1), 11p11-12 (EXT2), and 19p (EXT3). Constitutional chromosomal microdeletions of 8q24.1 and 11p11-12 are features of the Langer-Giedion and DEFECT-11 syndromes, respectively. Cytogenetic studies of osteochondroma are rare. METHODS: Cytogenetic analysis was performed on 34 osteochondroma specimens from 22 patients with sporadic lesions and 4 patients with HME utilizing standard methodologies. Fluorescence in situ hybridization with chromosome specific probes was performed on three cases to define structural rearrangements further. RESULTS: Clonal abnormalities were detected in ten cases. Notably, deletion of 11p11-13 was observed in one case (a sporadic tumor) and loss or rearrangement of 8q22-24.1 in eight cases (seven sporadic and one hereditary tumor). CONCLUSIONS: These findings: 1) confirm previous observations of 8q24.1 karyotypic anomalies in sporadic osteochondroma, 2) reveal the presence of somatic chromosomal anomalies in hereditary osteochondromata, 3) suggest that similar to hereditary lesions, sporadic osteochondromas also are genetically heterogeneic (involvement of both 8q24.1 and 11p11-12), and 4) support the hypothesis that loss or mutation of EXT1 and EXT2, two putative tumor suppressor genes, may be important in the pathogenesis of sporadic as well as hereditary osteochondromata.

Characterization of a variant SYT-SSX1 synovial sarcoma fusion transcript.

Synovial sarcoma is characterized cytogenetically by an X;18 translocation [t (X;18) (p11;q11)] that results in the fusion of the SYT gene from chromosome 18 to either of two highly homologous genes at Xp11, SSX1 or SSX2. Heterogeneity within the synovial sarcoma fusion junctions is rare. Examination of a primary monophasic synovial sarcoma for an SYT-SSX fusion transcript by reverse-transcription polymerase chain reaction revealed a smaller-than-expected product. Direct sequencing of the product disclosed a novel SYT-SSX1 fusion transcript that contained an additional 51 bp of normal SSX1 sequence but lost 135 bp of the SYT sequence. Detection of synovial sarcoma hybrid transcripts is useful for diagnosis and should include the recognition of distinct variants.