Toru Motoi

Identification of a Novel, Recurrent HEY1-NCOA2 Fusion in Mesenchymal Chondrosarcoma based on a Genome-wide Screen of Exon-level Expression Data

Cancer gene fusions that encode a chimeric protein are often characterized by an intragenic discontinuity in the RNA\expression levels of the exons that are 5′ or 3′ to the fusion point in one or both of the fusion partners due to differences in the levels of activation of their respective promoters. Based on this, we developed an unbiased, genome‐wide bioinformatic screen for gene fusions using Affymetrix Exon array expression data. Using a training set of 46 samples with different known gene fusions, we developed a data analysis pipeline, the “Fusion Score (FS) model”, to score and rank genes for intragenic changes in expression. In a separate discovery set of 41 tumor samples with possible unknown gene fusions, the FS model generated a list of 552 candidate genes. The transcription factor gene NCOA2 was one of the candidates identified in a mesenchymal chondrosarcoma. A novel HEY1‐NCOA2 fusion was identified by 5′ RACE, representing an in‐frame fusion of HEY1 exon 4 to NCOA2 exon 13. RT‐PCR or FISH evidence of this HEY1‐NCOA2 fusion was present in all additional mesenchymal chondrosarcomas tested with a definitive histologic diagnosis and adequate material for analysis (n = 9) but was absent in 15 samples of other subtypes of chondrosarcomas. We also identified a NUP107‐LGR5 fusion in a dedifferentiated liposarcoma but analysis of 17 additional samples did not confirm it as a recurrent event in this sarcoma type. The novel HEY1‐NCOA2 fusion appears to be the defining and diagnostic gene fusion in mesenchymal chondrosarcomas.

Immunohistochemical analysis of MDM2 and CDK4 distinguishes low-grade osteosarcoma from benign mimics.

Parosteal osteosarcoma and low-grade central osteosarcoma are two types of low-grade osteosarcoma that show similar clinical behaviors, histological features, and genetic background (ie, amplified sequences of 12q13–15, including MDM2 and CDK4). Low-grade osteosarcoma is often confused with benign lesions, and ancillary techniques to enhance diagnostic accuracy have been awaited. This study explores the use of MDM2 and CDK4 immunohistochemistry for the histological diagnosis of low-grade osteosarcoma. We studied 23 cases of low-grade osteosarcoma from 21 patients (parosteal osteosarcoma (n=14), low-grade central osteosarcoma (n=9)) and 40 cases of benign histological mimics (myositis ossificans (n=11), fibrous dysplasia (n=14), osteochondroma (n=6), desmoplastic fibroma (n=1), florid reactive periostitis (n=4), Noras lesion (n=3), and turret exostosis (n=1)). Low-grade osteosarcoma labeled for MDM2 in 16 cases (70%) and for CDK4 in 20 cases (87%). All low-grade osteosarcomas expressed one or both markers (100%), with 13 cases (57%) expressing both. Staining pattern was diffuse in most cases, and the majority expressed moderate or strong intensity for either antibody. MDM2/CDK4 immunostaining was shown irrespective of low-grade osteosarcoma histological subtype. In contrast, only 1 Noras lesion out of the 40 miscellaneous benign processes showed immunoreactivity for MDM2 or CDK4. The combination of these two markers thus shows 100% sensitivity and 97.5% specificity for the diagnosis of low-grade osteosarcoma. MDM2 and CDK4 immunostains therefore reliably distinguish low-grade osteosarcoma from benign histological mimics, and their combination may serve as a useful adjunct in this difficult differential diagnosis.

Retroperitoneal liposarcoma with combined well-differentiated and myxoid malignant fibrous histiocytoma-like myxoid areas.

To broaden the knowledge of myxoid morphology in liposarcoma, eight cases of unusual liposarcoma with combined well-differentiated and myxoid malignant fibrous histiocytoma (MFH)-like myxoid areas are reported. The tumors arose as huge retroperitoneal masses in elderly patients, except for one that occurred in the spermatic cord. Three cases had local recurrences, and one of the seven patients who were followed up had died of the tumor. Grossly, the tumors were mostly confluent and multinodular and showed a glistening myxoid appearance in variable proportions, which merged gradually into or were juxtaposed to yellow fatty or sclerotic whitish areas. Microscopically, in addition to areas of well-differentiated lipoma-like or sclerosing liposarcoma, all the tumors contained myxoid portions characterized by scattered multinucleated or bizarre giant cells and a prominent plexiform vascular pattern that resembled myxoid MFH or myxofibrosarcoma. The myxoid areas were associated with discernible lipogenesis. High-grade dedifferentiation was present in one tumor. Cytogenetically, in one case, the myxoid lesion had nonrandom chromosomal aberrations, such as ring and marker chromosomes, characteristic of a well-differentiated variant of liposarcoma. In a nested reverse transcription-polymerase chain reaction analysis using archival paraffin-embedded tissue, it was seen that none of the eight tumors with myxoid MFH-like features had TLS/FUS-CHOP fusion transcripts characteristic of myxoid and round cell liposarcomas. These clinicopathologic and molecular features suggest that the current myxoid tumors are more closely related to well-differentiated liposarcoma rather than to ordinary myxoid liposarcoma despite their unequivocal myxoid morphology. Missense point mutations of the p53 gene were detected in two (25%) cases by single-strand conformation polymorphism and sequence analyses. Immunohistochemical expressions of p53 and mdm2 were observed in 75% of the cases, in which immunoreactive tumor cells were seen more often in the myxoid MFH-like areas. Thus, altered p53 pathways, such as p53 gene mutation and mdm2-mediated inactivation of p53, may play a pathogenetic role in this form of tumor progression showing myxoid MFH-like morphology in liposarcoma, as has been suggested in dedifferentiated liposarcoma.

MDM2 and CDK4 immunohistochemical coexpression in high-grade osteosarcoma: correlation with a dedifferentiated subtype.

Low-grade osteosarcomas comprise a distinct subset of osteosarcomas. They may occasionally dedifferentiate into high-grade tumors, typically in the form of high-grade osteosarcoma, which are histologically indistinguishable from conventional osteosarcomas. MDM2 and CDK4 are often amplified in low-grade osteosarcomas and their dedifferentiated counterparts, and the encoded proteins are accordingly overexpressed. As MDM2/CDK4 expression was reportedly rare in conventional osteosarcoma, we hypothesized that these markers may help separate dedifferentiated osteosarcoma from the conventional type. To test this, we performed MDM2 and CDK4 immunohistochemistry on 81 primary and 26 recurrent/metastatic high-grade osteosarcomas and correlated these data with the histology of the primary resection material, with particular attention to the potential presence of any coexisting low-grade osteosarcomatous components. MDM2 and CDK4 coexpression was identified in 7 cases, and on careful histologic review 6 of them were discovered to contain foci of coexisting low-grade elements. One case was a known dedifferentiated parosteal osteosarcoma, and the remaining 5 cases were newly identified dedifferentiated osteosarcomas in which the limited low-grade components were originally unrecognized. An additional 11 cases expressed either marker alone, whereas the remaining 89 cases were negative for both markers; no resection material from these 100 cases presented with a low-grade component. MDM2/CDK4 gene amplification status, determined by quantitative polymerase chain reaction in selected cases, was largely concordant with immunoexpression. Our data suggest that MDM2 and CDK4 coexpression in high-grade osteosarcomas is sensitive and specific to those that progressed from low-grade osteosarcomas, and immunohistochemistry may help identify this dedifferentiated subgroup to facilitate accurate subclassification.

CIC-rearranged Sarcomas: A Study of 20 Cases and Comparisons With Ewing Sarcomas.

The CIC gene rearrangement exists in a subset of small round cell sarcomas. As the nosologic relationship of these sarcomas to Ewing sarcomas remains undetermined, we examined 20 CIC-rearranged sarcomas to compare their clinicopathologic features with those of Ewing sarcomas. The CIC-rearranged sarcomas were from a group of 14 men and 6 women with a median age of 24.5 years. The primary tumor sites included the limbs, trunk wall, internal trunk, lung, cerebrum, and pharynx. A comparison of the demographic and clinical characteristics of the 20 patients with CIC-rearranged sarcomas with those of the 53 near-consecutive patients with EWSR1-rarranged Ewing sarcomas showed that there were no differences with respect to their ages and sexes. Although none of the CIC-rearranged sarcomas arose in the bone, 40% of the Ewing sarcomas primarily affected the skeleton. The overall survival of patients with Ewing sarcomas was significantly better than that for patients with CIC-rearranged sarcomas. A histologic comparison of the CIC-rearranged sarcomas with 20 EWSR1-rearranged Ewing sarcomas showed significantly higher degrees of lobulation, nuclear pleomorphism, the prominence of the nucleoli, spindle cell elements, and myxoid changes in the CIC-rearranged sarcomas. Distinguishing immunohistochemical features included heterogenous CD99 reactivity, nuclear WT1 expression, and calretinin expression in the CIC-rearranged sarcomas and NKX2.2 expression in the Ewing sarcomas. CIC-rearranged sarcomas are distinct from Ewing sarcomas clinically, morphologically, and immunohistochemically, and they should be considered a separate entity rather than being grouped within the same family of tumors.The CIC gene rearrangement exists in a subset of small round cell sarcomas. As the nosologic relationship of these sarcomas to Ewing sarcomas remains undetermined, we examined 20 CIC-rearranged sarcomas to compare their clinicopathologic features with those of Ewing sarcomas. The CIC-rearranged sarcomas were from a group of 14 men and 6 women with a median age of 24.5 years. The primary tumor sites included the limbs, trunk wall, internal trunk, lung, cerebrum, and pharynx. A comparison of the demographic and clinical characteristics of the 20 patients with CIC-rearranged sarcomas with those of the 53 near-consecutive patients with EWSR1-rarranged Ewing sarcomas showed that there were no differences with respect to their ages and sexes. Although none of the CIC-rearranged sarcomas arose in the bone, 40% of the Ewing sarcomas primarily affected the skeleton. The overall survival of patients with Ewing sarcomas was significantly better than that for patients with CIC-rearranged sarcomas. A histologic comparison of the CIC-rearranged sarcomas with 20 EWSR1-rearranged Ewing sarcomas showed significantly higher degrees of lobulation, nuclear pleomorphism, the prominence of the nucleoli, spindle cell elements, and myxoid changes in the CIC-rearranged sarcomas. Distinguishing immunohistochemical features included heterogenous CD99 reactivity, nuclear WT1 expression, and calretinin expression in the CIC-rearranged sarcomas and NKX2.2 expression in the Ewing sarcomas. CIC-rearranged sarcomas are distinct from Ewing sarcomas clinically, morphologically, and immunohistochemically, and they should be considered a separate entity rather than being grouped within the same family of tumors.

The EWSR1/NR4A3 fusion protein of extraskeletal myxoid chondrosarcoma activates the PPARG nuclear receptor gene

The NR4A3 nuclear receptor is implicated in the development of extraskeletal myxoid chondrosarcoma (EMC), primitive sarcoma unrelated to conventional chondrosarcomas, through a specific fusion with EWSR1 resulting in an aberrant fusion protein that is thought to disrupt the transcriptional regulation of specific target genes. We performed an expression microarray analysis of EMC tumours expressing the EWSR1/NR4A3 fusion protein, comparing their expression profiles to those of other sarcoma types. We thereby identified a set of genes significantly overexpressed in EMC relative to other sarcomas, including PPARG and NDRG2. Western blot or immunohistochemical analyses confirm that PPARG and NDRG2 are expressed in tumours positive for EWSR1/NR4A3. Bioinformatic analysis identified a DNA response element for EWSR1/NR4A3 in the PPARG promoter, and band‐shift experiments and transient transfections indicate that EWSR1/NR4A3 can activate transcription through this element. Western blots further show that an isoform of the native NR4A3 receptor lacking the C‐terminal domain is very highly expressed in tumours positive for EWSR1/NR4A3, and co‐transfections of this isoform along with EWSR1/NR4A3 indicate that it may negatively regulate the activity of the fusion protein on the PPARG promoter. These results suggest that the overall expression of PPARG in EMC may be regulated in part by the balance between EWSR1/NR4A3 and NR4A3, and that PPARG may play a crucial role in the development of these tumours. The specific up‐regulation of PPARG by EWSR1/NR4A3 may also have potential therapeutic implications. Copyright

TLS-CHOP represses miR-486 expression, inducing upregulation of a metastasis regulator PAI-1 in human myxoid liposarcoma.

Myxoid liposarcomas (MLSs) are characterized by t(12;16)(q13;p11) translocation and expression of TLS-CHOP chimeric oncoprotein. However, the molecular functions of TLS-CHOP have not been fully understood. On the other hand, microRNAs (miRNAs) comprise an abundant class of endogenous small non-coding RNAs that negatively regulate the expression of their target genes, and are involved in many biological processes. It is now evident that dysregulation of miRNAs is an important step in the development of many cancers. To our knowledge, however, there have been no reports of the miRNAs involved in MLS tumorigenesis and development. In this study, we have found that miR-486 expression was repressed in TLS-CHOP-expressed NIH3T3 fibroblasts and MLS tissues, and exogenous overexpression of miR-486 repressed growth of MLS cells. Thus, downregulation of miR-486 may be an important process for MLS. In addition, we have identified plasminogen activator inhibitor-1 (PAI-1) as a novel target gene of miR-486. PAI-1 is a unique type of serine protease inhibitor and is known to be one of the key regulators of tumor invasion and metastasis. Furthermore, knockdown of PAI-1 by a specific small interfering RNA (siRNA) inhibited growth of MLS cells, suggesting that increased expression of PAI-1 by miR-486 repression is critical for survival of MLS cells. Collectively, these results suggest a novel essential molecular mechanism that TLS-CHOP activates PAI-1 expression by repression of miR-486 expression in MLS tumorigenesis and development.

Well-differentiated liposarcoma with low-grade osteosarcomatous component: an underrecognized variant.

Mature bone formation in well-differentiated liposarcoma and dedifferentiated liposarcoma has been described as a reactive or “metaplastic” change in most reports, and its neoplastic nature has not been widely appreciated. We herein describe 9 cases of well-differentiated/dedifferentiated liposarcoma with distinct areas of fibroosseous tissue histologically indistinguishable from low-grade osteosarcomas, that is, parosteal osteosarcoma or low-grade central osteosarcoma. The tumors affected middle-aged to elderly patients, and occurred in the retroperitoneum and deep soft tissue of the extremities without connection to the skeletal system. Grossly, all the tumors showed biphasic appearance with lipogenic and osteogenic area, the latter representing 5% to 50% of the total tumor volume. Histologically, the lipogenic component exhibited typical histology of well-differentiated liposarcoma, whereas the osteogenic area consisted of fibroosseous tissue with numerous mature neoplastic bone trabeculae largely lacking osteoblastic rimming, with intervening fascicles of spindle cell proliferation showing low nuclear grade. All samples were positive for MDM2 and/or CDK4 on immunohistochemical analysis; the antibodies stained many osteocytes, indicating that the bone is neoplastic rather than reactive. Three cases showed high-grade osteosarcomatous transformation juxtaposed to the low-grade osteosarcomatous component, reminiscent of the “dedifferentiation” phenomenon of skeletal low-grade osteosarcoma. Follow-up revealed local recurrence in 4 cases, but no distant metastases were documented. Recognition of this earlier underappreciated subtype of well-differentiated/dedifferentiated liposarcoma is important, because the fibroosseous component may seem so bland that it may be confused with benign metaplasia such as myositis ossificans, or conversely, the lipomatous component may be inconspicuous that it may be dismissed as normal fat, and such misinterpretation may potentially result in suboptimal treatment.

Synovial sarcoma of the head and neck: Rare case of cervical metastasis

Synovial sarcoma is a rarely encountered soft tissue sarcoma. Surgery with a wide surgical margin is the treatment of choice. However, there is no consensus on the treatment of head and neck synovial sarcoma in patients with cervical metastasis.

Osteomalacia caused by skull base tumors: report of 2 cases.

BACKGROUND AND IMPORTANCE:Tumor-induced osteomalacia (TIO) is an uncommon paraneoplastic syndrome rarely encountered in neurosurgical practice. We report on 2 cases of TIO caused by skull base tumors. Although the diagnosis of TIO is difficult to make and often is delayed because of the insidious nature of the symptoms, mostly systemic pain and weakness, it is curable once it is diagnosed and properly treated. CLINICAL PRESENTATION:Both patients presented with severe pain developing in the lower extremities and moving out to the entire body, as well as difficulty moving. They were diagnosed with TIO several years after onset. A high level of serum FGF23 was confirmed, and whole-body imaging studies demonstrated tumors in the middle and anterior cranial base, respectively. The patient with the anterior cranial base tumor had a history of hemorrhage into the frontal lobe and partial resection. En bloc resection of tumor with surrounding skull bone was performed. The histological diagnosis for both cases was phosphaturic mesenchymal tumor, mixed connective tissue variant. CONCLUSION:The level of FGF23 normalized immediately after surgery. Both patients experienced a dramatic relief of pain and recovery of muscle power. Although reports of osteomalacia caused by tumors in the neurosurgical field are extremely rare in the literature, its true incidence is unknown. We emphasize the importance of recognition of this syndrome and recommend total resection of tumors when possible.