Masanori Hisaoka

Detection of SYT-SSX Fusion Transcripts in Synovial Sarcoma by Reverse Transcription-Polymerase Chain Reaction Using Archival Paraffin-Embedded Tissues

The reciprocal translocation t(X;18)(p11;q11) is known to be highly characteristic of synovial sarcoma, and its consequence, an SYT-SSX fusion gene, is expected to be a diagnostic molecular marker. In this study, we conducted a reverse transcription-polymerase chain reaction-based assay to detect the SYT-SSX fusion gene transcripts using archival formalin-fixed, paraffin-embedded tumor specimens from a series of 32 synovial sarcoma cases including 6 tumors found in unusual anatomical sites. The SYT-SSX fusion transcripts could be detected in 30 of 32 paraffin-embedded specimens (94%). A subsequent sequence analysis using the polymerase chain reaction products confirmed that the detected messages were derived from either the SYT-SSX1 (22 cases) or SYT-SSX2 (8 cases) fusion gene. Of 23 SYT-SSX-positive monophasic tumors, 16 tumors had an SYT-SSX1 fusion transcript. Fusion transcripts were detectable in all the 7 biphasic tumors analysed, one of which had an SYT-SSX2 fusion transcript. All of the six tumors at unusual locations (lung; 3, metastasis to the abdominal cavity from a tumor of retroperitoneal origin; 1, sacral region; 1, iliopsoas muscle; 1) contained detectable messages. Our results indicate that this molecular assay can be applied to archival formalin-fixed, paraffin-embedded tumor tissues as a feasible and reliable molecular technique for the diagnosis of synovial sarcoma.

Overexpression of the Hepatocyte Growth Factor (HGF) Receptor (Met) and Presence of a Truncated and Activated Intracellular HGF Receptor Fragment in Locally Aggressive/Malignant Human Musculoskeletal Tumors

Enhanced hepatocyte growth factor (HGF) receptor (Met) signaling has been suggested to play an important role in the development and progression of various epithelial and nonepithelial tumors. N-terminally truncated forms of the HGF receptor have been shown to be constitutively activated and tumorigenic in animal experiments. In the present study, 102 benign and malignant human musculoskeletal tumors were examined for expression of the HGF receptor by Western blotting and/or immunohistochemistry. A clear predominance of HGF receptor expression was seen in malignant as compared to benign tumors (Western blotting, P < 0.001; immunohistochemistry, P < 0.02). For the first time we show HGF receptor expression in the following four tumor types: dermatofibrosarcoma protuberans, clear cell sarcoma of tendons, malignant primitive neuroectodermal tumor, and benign fibrous histiocytoma. In three cases of sarcoma with high HGF receptor expression by Western blotting, we found indications of a short 85-kd N-terminally truncated HGF receptor that was tyrosine phosphorylated and located in the cytoplasm. Although fragments of this length were seen in 18 of 65 tumors, most were not tyrosine-phosphorylated. Northern blotting revealed only the 7.5-kb full-length HGF receptor transcript, suggesting that the 85-kd fragment is generated by an alternative initiation of translation or by proteolytic cleavage. Southern blotting detected no amplification of the Hgfr/Met gene in the 35 tumors examined, in contrast to our recent report of Hgfr/Met gene amplification in 7, 12-dimethylbenz(a)anthracene (DMBA)-induced rat sarcomas. The present data suggest that the locally aggressive and malignant properties of human mesenchymal tumors maybe related, in part, to high levels of full-length HGF receptors, and in some cases to the occurrence of N-terminally truncated HGF receptors, activated independently of HGF.

Dermatofibrosarcoma protuberans with fibrosarcomatous areas. Molecular abnormalities of the p53 pathway in fibrosarcomatous transformation of dermatofibrosarcoma protuberans.

Abstractu2002Fibrosarcomatous (FS) change in a rare, but well-known phenomenon encountered in dermatofibrosarcoma protuberans (DFSP), and an increased chance in an adverse outcome has been suggested in patients with DFSP having FS areas (DFSP-FS). As altered p53 pathway has been suggested as having a potential role in tumour progression, we analysed the p53 gene and p53 protein together with the p53-related protein mdm2 and p21Wafl in 5 cases of DFSP-FS and 13 of DFSP to ascertain whether the p53 pathway correlates to the fibrosarcomatous transformation of DFSP. Three of the five DFSP-FSs overexpressed p53 protein immunohistochemically, and one of them had a ”missense” mutation of the p53 gene without immunohistochemical overexpression of mdm2 or p21Wafl. The other two DFSP-FSs with p53 overexpression demonstrated increased labelling indices of both mdm2 and p21Wafl. The three DFSP-FS patients with overexpression of p53 protein had frequent local recurrences, ranging from 3 to 5 in number with increasingly short intervals (mean 4.5 years), while one of the other two had no recurrences and the other, only one. None of the 13 DFSPs showed any alterations in the p53 gene or overexpressions of p53, mdm2 and p21Wafl, except for one DFSP having a ”silent” mutation of the p53 gene. Three DFSPs had local recurrences once or twice with longer intervals to recurrence (mean 10.3 years). Although the number of cases examined is limited, the results suggest that alterations in the p53 pathway, such as overexpression of p53 protein by a mutated gene and mdm2 overexpression, are involved in fibrosarcomatous transformation in a subset of fibrohistiocytic tumours and possibly correlated with its more locally aggressive behaviour than that without p53 alterations or ordinary DFSP.

Clinicopathological and interphase cytogenetic analysis of desmoid tumours.

Recurrence of desmoid tumours is difficult to predict from only histological findings. In this study, immunohistochemistry for counting stromal blood vessels and proliferative activity, DNA flow cytometry, and interphase cytogenetic analysis of chromosome 8 by fluorescence in‐situ hybridization (FISH) were performed to assess the correlation between their parameters and the recurrence of desmoid tumours.

Peripheral primitive neuroectodermal tumour of the lung: report of two cases.

We report two unusual cases of intrapulmonary small round cell tumour with evidence that they are primitive neuroectodermal tumours.

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.

Peripheral primitive neuroectodermal tumour with ganglioneuroma-like areas arising in the cauda equina.

Abstractu2002Peripheral primitive neuroectodermal tumour (pPNET or peripheral neuroepithelioma) is one of the malignant small round cell tumours of peripheral nerves, soft tissues and bones, but rarely originates in the spinal canal. We report an example of pPNET arising in the cauda equina of a 14-year-old Japanese boy. At surgery, a well-demarcated tumour measuring 2×4u2005cm in diameter and involving one of the nerve roots of the cauda equina was located within the intradural space with no evidence of extradural extension. Microscopically the tumour was made up of sheets of closely packed small round cells, associated with ganglioneuroma-like islands. Immunohistochemically, the small round tumour cells were intensely positive for neuron-specific enolase (NSE), an MIC2 gene product (O13) and β2-microglobulin, whereas the foci with ganglion cell-like cells reacted positively to NSE, synaptophysin and β2-microglobulin but were negative for O13. A chimeric transcript of the EWS/FLI-1 fusion gene detected by a nested reverse transcriptase–polymerase chain reaction using formalin-fixed paraffin-embedded tissue justified the diagnosis of pPNET. Only 6 cases of PNET in the cauda equina have been described in the literature, and this is the first case of a pPNET with ganglioneuroma-like areas. This finding suggests that the primitive tumour cells of pPNET may respond to unknown inductive effects and express a ganglion cell-like morphology.

Molecular detection of EWS-FLI1 chimeric transcripts in Ewing family tumors by nested reverse transcription-polymerase chain reaction : application to archival paraffin-embedded tumor tissues

Chromosomal translocations generating unique chimeric genes are highly characteristic of specific sarcomas, and their use as diagnostic markers has been suggested. From a diagnostic pathologic point of view, detection of such cytogenetic or molecular aberrations applicable to routinely processed archival tissue specimens is considered a powerful tool for tumor diagnosis. To assess the feasibility and reliability of the molecular detection of the transcript originating from the chimeric gene in paraffin‐embedded tumor specimens, we performed a nested reverse transcription‐polymerase chain reaction (RT‐PCR)‐based assay to detect the EWS‐FLI1 chimeric message in a series of Ewing family tumors. Of 24 paraffin‐embedded tumor specimens from 23 cases analyzed, the chimeric message was detectable in 20 (83%) specimens from 20 cases (87%) by this nested RT‐PCR assay, whereas none of 7 small round cell tumors not from this family (3 alveolar rhabdomyosarcomas, 2 neuroblastomas, 2 malignant lymphomas) showed detectable chimeric messages. In the sequence analysis of the PCR products, the amplified chimeric messages contained the junctions between exon 7 of the EWS gene and any one of exons 5, 6 and 8 of the FLI1 gene. The detection process was usually completed within 3 days, except for the subseqent sequence analysis. Our results endorse the use of this molecular assay as an ancillary technique in the diagnosis of Ewing family tumors using paraffin‐embedded material.

Dermatofibrosarcoma protuberans and its fibrosarcomatous variant with areas of myoid differentiation: a report of three cases

We describe three examples of dermatofibrosarcoma protuberans which demonstrated focal myofibroblastic differentiation, and discuss the nature of myofibroblastic differentiation.

Dedifferentiated liposarcoma with an inflammatory malignant fibrous histiocytoma‐like component presenting a leukemoid reaction

A rare case of dedifferentiated llposarcoma (well‐differentiated llposarcoma with an inflammatory mallgnant fibrous histiocytoma (MFH)‐like anaplastlc component) occurrtng in a 69‐year‐old male is presented. The patient had noticed a dull pain in his left loin and thigh for about 1 month. Computed tomography examination revealed a low‐density mass lesion, measuring about 6 cm In diameter, in the left lliopsoas muscie, and It was surgically removed. Grossly, the lesion was composed of an encapsulated, soft, whitish mass and an adjacent, well‐demarcated, yellowish hard nodule, measuring about 2.5 cm In diameter. Microscopically, both lesions showed features of an inflammatory variant of MFH and a sclerosing type of well‐differentiated liposarcoma, respectively. To our knowledge, only two cases of dedifferentiated liposarcoma combined with inflammatory MFH as a dedifferentiated component have been recorded in the literature. The salient feature of the present case is a systemic inflammatory reaction, as shown by prominent leukocytosis (up to 73 900/mm3) and the elevated serum value of C reactive protein (up to 26.0 mg/dL), which were transiently reduced after surgery. The Inflammatory reaction was suggested to be induced by cytokines, such as granulocyte colony‐stimulating factor and interleukin‐6, which were probably produced by the tumor cells in the present case, because the elevated serum values of those cytokines were decreased after surgery.