Toshifumi Takakuwa

Immunocytochemical and ultrastructural studies of the histogenesis of Ewing's sarcoma and putatively related tumors

The histogenesis of Ewings sarcoma (EW) and extraskeletal Ewings sarcoma (EEW) is still disputable. Their relationship to the so‐called Askins tumor, neuroectodermal tumor of bone, and peripheral neuroblastoma remains to be established. In an attempt to clarify these points, immunocytochemical and ultrastructural studies were done on tissues from 14 cases of EW, 4 cases of EEW, and 9 cases of primitive neuroectodermal tumor (PNET) and compared with neuroblastoma and olfactory neuroblastoma. Six tumors categorized initially as EW and EEW on biopsy, turned out to be PNET by extensive histologic and/or ultrastructural observations. Abundant glycogen was recognized not only in 16 of 18 cases of EW and EEW, but also in seven of nine cases of PNET. Fine fibrillar cell processes were seen between tumor cells, at least in limited areas even in cases of EW and EEW. Immunocytochemically, neuron‐specific enolase (NSE), neuroblastoma cell surface antigen (NBCA), neuron cell surface antigen (NCSA), and neurofilament (NF) were demonstrated not only in neuroblastoma, but also frequently in cases of EW, EEW, and PNET. The results seem to suggest that EW and EEW represent the most immature forms of neuroectodermal tumor. Electron microscopic study showed predominantly primitive cells with occasional areas of cell processes, neurosecretory granules, and microtubules, suggesting a neuroectodermal origin.

PERINEURIAL CELL TUMOR AND THE SIGNIFICANCE OF THE PERINEURIAL CELLS IN NEUROFIBROMA

The authors attempted to clarify the exact cell components of neurofibroma by immunohistochemical and ultrastructural studies. Materials were randomly selected, 40 cases of neurilemoma and neurofibroma (‐tosis) in addition to 2 cases of tumors composed exclusively of perineurial cells and three cases of normal peripheral nerve. The applied markers included antisera of S‐100 protein for Schwann cells, blood coagulation factor XIIIa for endoneurial fibroblasts or perineurial cells, and laminin and collagen type IV for the basement membrane. S‐100 protein was demonstrated only in normal or neoplastic Schwann cells, but not in perineurial cells. On the other hand, factor XIIIa was often recognized in endoneurial fibroblasts and perineurial cells, but not in Schwann cells. Neurofibroma was basically composed of a mixture of Schwann cells, perineurial cells, and endoneurial fibroblasts, the population of each type of cell differing according to the case and area within a given tumor. Perineurial cell tumor exclusively composed of perineurial cells, though rare, appears to be a definite entity, and its characteristic histological and ultrastructural features were described.

Involvement of cell‐mediated killing in apoptosis in histiocytic necrotizing lymphadenitis (Kikuchi‐Fujimoto disease)

Histiocytic necrotizing lymphadenitis, also called Kikuchi‐Fujimoto (KF) disease, is a benign disorder characterized histologically by paracortical necrotic foci surrounded by histiocytic aggregates. We analysed affected lymph node tissues from 34 patients with the disease in an attempt to elucidate its histogenesis. The ‘necrotizing’ cells showed typical apoptotic changes, including cell shrinkage and condensed and fragmented nuclei. Apoptotic bodies with a peculiar ultrastructure were demonstrated, and DNA fragmentation was detected in these cells by in situ end labelling. Immunostaining for the apoptosis‐regulating proteins bcl‐2, bax, c‐myc and p53 failed to show their involvement in KF disease. However, perforin, a killer cell‐specific cytolytic protein essential for provoking apoptosis in target cells, was found to be expressed abundantly by the infiltrating cells, which were thought to be cytotoxic T‐lymphocytes. Perforin‐expressing cells were present in the apoptotic foci of 28 of the 34 patients (82.4%). Virtually no cells containing perforin granules were present in non‐pathological regions, lymph node tissues from control subjects with reactive or tuberculous lymphadenitis or those from patients with KF disease with negligible apoptosis. Therefore, the ‘necrosis’ associated with KF disease appears to be attributable to trans apoptotic death of the killer cell target in the affected nodes. We propose that KF disease should be called apoptotic lymphadenitis.

PRIMITIVE NEUROECTODERMAL TUMORS OF BONE AND SOFT TISSUE : WITH REFERENCE TO HISTOLOGIC DIFFERENTIATION IN PRIMARY OR METASTATIC FOCI

Primitive neuroectodermal tumors (PNET) of the bone and soft tissue were reviewed by immunohistochemistry and partly by morphometry, focusing particularly on histologic changes in recurrent or metastatic foci, in order to elucidate their probable histogenetic relationship with Ewings sarcoma (ES) and its extraskeletal counterpart (EES). Eleven cases of bone tumor (average patient age; 15.1 yr) and 12 cases of soft tissue tumor (average patient age; 22.1 yr) which disclosed unequivocal Homer Wright rosettes and/or at least foci of ganglion cell differentiation either in a given primary tumor or metastatic (or recurrent) foci were selected from small round cell tumors primarily categorized as ES or EES. Most of the cases for which follow up biopsy samples were available disclosed prominent Homer Wright rosettes in the metastases, whereas the primary tumors showed features of ES and lacked rosettes. In only one case, Homer‐Wright rosettes were absent in the metastatic tumor. Most cases had been treated by combined intensive chemotherapy and radiotherapy, which might have influenced cell differentiation. Neural markers (neuron‐specific enolase, neurofilament protein and others) were positive in most cases. Three cases with otherwise typical histologic features of ES or EES showed minute foci of ganglion cell differentiation, as confirmed by morphometry and neural markers. These results suggest that ES (or EES) and PNET are histo‐genetically related, but represent different stages of cell differentiation.

ULTRASTUCTURE OF CARTILAGINOUS TUMORS AND S‐100 PROTEIN IN THE TUMORS: With Reference to the Histogenesis of Chondroblastoma, Chondromyxoid Fibroma and Mesenchymal Chondrosarcoma

Twelve cartilaginous tumors were studied by electron microscopy and the presence of S‐100 protein was studied immunohistochemically in order to clarify the cell origin of chondroblastoma, chondromyxoid fibroma, and mesenchymal chondrosarcoma. Three chondroblastomas were characterized by round or ovoid tumor cells with some cytoplasmic processes, well‐developed organelles and thick fibrous laminae in the nuclear membrane, occasional multinucleated giant cells and scanty chondroid matrix. S‐100 protein was demonstrated in the tumor cells and some multinucleated giant cells. Two chondromyxoid fibromas revealed tumor cells of varied shapes with character istic cartilaginous differentiation and abundant chondroid matrix. Spindle tumor cells showed the ultrastructural features of cartilage cells rather than of fibroblasts and S‐100 protein was also demontratrated in their cytoplasm. Chondroblastoma and chondomyxoid fibroma were considered to arise from chondrocytes. Mesenchymal chondrosarcoma ultrastructurally exhibited round tumor cells with cartilaginous nature in cartilage islands. Poorly‐differentiated portions were composed of primitive round or elongated cells with occasionally admixed round cells with ultrastructural features of cartilaginous differentiation. S‐100 protein was demonstrated in the cells in cartilage islands and in single cells admixed in poorly‐differentiated portions. These results support the hypothesis of primitve mesenchymal origin with a tendency to differentiate toward cartilage cells. ACTA PATHOL. JPN. 34: 1285–1300, 1984.

Absence of Nuclear p16 From Epstein-Barr Virus-Associated Undifferentiated Nasopharyngeal Carcinomas

Objective: Epstein‐Barr virus (EBV) is detected in the majority of undifferentiated nasopharyngeal carcinomas (UNPCs, World Health Organization type III). However, the exact mechanism involved in the carcinogenesis of EBV‐associated UNPCs remains to be elucidated. An important unresolved question is: how is the normal cell cycle deregulated during EBV‐associated UNPC development? The p16CDKN2 gene encodes a nuclear protein, p16, which inhibits the D‐type cyclin/cyclin‐dependent kinase complexes that phosphorylate the retinoblastoma gene product (pRb), thus blocking G1 cell cycle progression. The objective of this study was to determine whether p16 absence is involved in the development of EBV‐associated UNPCs.

Demonstration of apoptosis in neuroblastoma and its relationship to tumour regression.

The in vivo occurrence of apoptosis in neuroblastomas was investigated. Histologically, a number of tumour cells showed typical apoptotic changes, including cell shrinkage, condensed and fragmented nuclei, eosinophilic cytoplasm, and absence of the inflammatory response. These cells coincided closely with the so-called karyorrhectic cells. An electrophoretic DNA ladder, a functional hallmark of apoptosis, was demonstrated in four of six tumours, and DNA fragmentation was detected in situ by terminal deoxytransferase-mediated nick end-labelling in 26 of 35 tumour specimens (74%). The labelled cell counts ranged from 5 to 62 per 5000 tumour cells (mean±SD: 15.0±14.5). Immunoperoxidase staining revealed that an apoptosis-suppressing protein, bcl-2, was expressed abundantly in advanced-stage tumours, whereas it was absent from karyorrhectic-apoptotic cells. Several tumours with the potential for spontaneous regression were bcl-2-deficient. Immunostaining of the Fas receptor for apoptosis demonstrated that the tumour cells expressed this molecule on their cell surfaces. Our results provide evidence of apoptosis in neuroblastomas and suggest that bcl-2 and the Fas receptor may play a role in its regulatory mechanisms.

PROLIFERATIVE MYOSITIS AND FASCIITIS: Report of Five Cases with an Ultrastructural and Immunohistochemical Study

Cases of proliferative myositis and fasciitis were studied immunohisto‐chemically and ultra structurally for further understanding of the nature of ganglion cell‐like giant cells. Blood coagulation factor XIIIa, fibronectin, myoglobin, myosin, CPK MM, and alpha‐1‐antichymotrypsin were detected in three cases of proliferative myositis and two cases of proliferative fasciitis by the avid in‐biotin‐peroxidase complex method. Factor XIIIa (a fibrin‐stabilizing factor) and flbronectin were strongly positive in the giant cells, but not in striated muscle fibers. A small quantity of myosin was demonstrated in the giant cells, but myoglobin and CPK MM were never demonstrated in these cells. No alpha‐1‐antichymotrypsin was demonstrated in the giant cells. One case of proliferative myositis showed ultrastructural features suggestive of fibroblast rather than muscle cell or histiocytic origin. Strongly positive factor XIIIa in the giant cells is suggestive of the fact that they are active fibroblasts.

Case report 263

A 13-year-old girl was admitted to the St. Marianna University Hospital on 26 February 1981 because of a gradually increasing focal swelling in the lower segment of the left thoracic cage, together with a history of pain in that region over a seven-month period. Radiological studies on admission showed a destructive, expanding lesion of the left 10th rib, associated with a large soft tissue mass. A thin outer bony shell of the 10th rib was apparent (Fig. 1).

Immunohistochemical expression of manganese superoxide dismutase in hepatocellular carcinoma, using a specific monoclonal antibody

The expression of manganese superoxide dismutase (Mn−SOD) was studied immunohistochemically, using a specific monoclonal antibody, in surgically resected hepatocellular carcinoma (HCC) and noncancerous tissues from 47 patients (2 with well-differentiated HCC, 36 with moderately differentiated HCC, 8 with poorly differentiated HCC, and 1 with undifferentiated carcinoma). Cancer cells in 44 patients (93.6%) were positive for Mn−SOD. The staining pattern of cancer cells was mostly homogeneous in well-differentiated HCC, whereas it was heterogeneous in poorly differentiated HCC. Moreover, strongly positive immunoreactivity was observed in noncancerous liver tissues in all patients, especially in normal hepatocytes surrounding HCC, regenerative small hepatocytes in the tumor boundary, and mononuclear inflammatory cells in the necroinflammatory lesions. The positive immunoreactivity for Mn−SOD in patients with HCC appears to reflect increased production of the enzyme protein.