Kunihiko Seki

Gene expression analysis of soft tissue sarcomas: characterization and reclassification of malignant fibrous histiocytoma

In soft tissue sarcomas, the diagnosis of malignant fibrous histiocytoma (MFH) has been a very controversial issue, and MFH is now considered to be reclassified into pleomorphic subtypes of other sarcomas. To characterize MFH genetically, we used an oligonucleotide microarray to analyze gene expression in 105 samples from 10 types of soft tissue tumors. Spindle cell and pleomorphic sarcomas, such as dedifferentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor (MPNST), fibrosarcoma and MFH, showed similar gene expression patterns compared to other tumors. Samples from those five sarcoma types could be classified into respective clusters based on gene expression by excluding MFH samples. We calculated distances between MFH samples and other five sarcoma types (dedifferentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, MPNST and fibrosarcoma) based on differentially expressed genes and evaluated similarities. Three of the 21 MFH samples showed marked similarities to one of the five sarcoma types, which were supported by histological findings. Although most of the remaining 18 MFH samples showed little or no histological resemblance to one of the five sarcoma types, 12 of them showed moderate similarities in terms of gene expression. These results explain the heterogeneity of MFH and show that the majority of MFHs could be reclassified into pleomorphic subtypes of other sarcomas. Taken together, gene expression profiling could be a useful tool to unveil the difference in the underlying molecular backgrounds, which leads to a rational taxonomy and diagnosis of a diverse group of soft tissue sarcomas.

Immunohistochemical detection of GLUT-1 can discriminate between reactive mesothelium and malignant mesothelioma

The separation of benign reactive mesothelium (RM) from malignant mesothelial proliferation can be a major challenge. A number of markers have been proposed, including epithelial membrane antigen, p53 protein, and P-glycoprotein. To date, however, no immunohistochemical marker that allows unequivocal discrimination of RM from malignant pleural mesothelioma (MPM) has been available. A family of glucose transporter isoforms (GLUT), of which GLUT-1 is a member, facilitate the entry of glucose into cells. GLUT-1 is largely undetectable by immunohistochemistry in normal epithelial tissues and benign tumors, but is expressed in a variety of malignancies. Thus, the expression of GLUT-1 appears to be a potential marker of malignant transformation. Recently, in fact, some studies have shown that GLUT-1 expression is useful for distinguishing benign from malignant lesions. The purpose of the present study was to evaluate the diagnostic utility of GLUT-1 expression for diagnostic differentiation between RM and MPM. Immunohistochemical staining for GLUT-1 was performed in 40 cases of RM, 48 cases of MPM, and 58 cases of lung carcinoma. Immunohistochemical GLUT-1 expression was seen in 40 of 40 (100%) MPMs, and in all cases the expression was demonstrated by linear plasma membrane staining, sometimes with cytoplasmic staining in addition. GLUT-1 expression was also observed in 56 out of 58 (96.5%) lung carcinomas. On the other hand, no RM cases were positive for GLUT-1. GLUT-1 is a sensitive and specific immunohistochemical marker enabling differential diagnosis of RM from MPM, whereas it cannot discriminate MPM from lung carcinoma.

Glut-1 expression and enhanced glucose metabolism are associated with tumour grade in bone and soft tissue sarcomas: a prospective evaluation by [18F]fluorodeoxyglucose positron emission tomography

PurposeThis study was conducted to investigate whether 18F-fluorodeoxyglucose (FDG) uptake, quantified by positron emission tomography (PET), correlates with histological variables including tumour grade, cell proliferation, cell cycle control integrity and glucose metabolism in patients with bone and soft tissue sarcomas.MethodsEighty-two patients clinically suspected of having a bone or soft tissue sarcoma underwent FDG PET within 1 week prior to operation and 63 patients (mean age 48 years, range 18–74 years) were enrolled in the complete analysis. We excluded 17 patients with pathologically confirmed benign tumours and two patients with uncontrolled diabetes or concomitant malignancy from data analysis. Maximum and average standardised uptake values (SUVs) of the primary lesion were compared with histological variables including tumour differentiation, the presence of necrosis, MIB-1 score, mitotic score, p53 overexpression, MIB-1 grade, mitotic grade and GLUT-1 expression.ResultsSignificant correlations were found between maximal and mean SUVs and MIB-1 grade, mitotic grade, MIB-1 score, tumour differentiation and mitotic score. The mean and maximal SUVs were significantly higher in tumours with p53 overexpression than in those without p53 overexpression (p<0.0001). GLUT-1-positive tumours had significantly higher mean (6.5±4.2 vs 1.1±0.2, p=0.006) and maximal SUVs (8.8±5.4 vs 1.7±0.5, p=0.005) than the GLUT-1-negative tumours. GLUT-1 intensity correlated positively with both mean (r=0.500, p<0.0001) and maximal SUVs (r=0.509, p<0.0001). Mu ltiple linear regression analysis showed a significant correlation between maximal SUV and MIB-1 grade (p<0.0001).ConclusionThe enhanced glucose metabolism, as determined by SUV, is a strong index of tumour grade in bone and soft tissue sarcomas.

Pfetin as a Prognostic Biomarker of Gastrointestinal Stromal Tumors Revealed by Proteomics

Purpose: We aimed to develop prognostic biomarkers for gastrointestinal stromal tumors (GIST) using a proteomic approach. Experimental Design: We examined the proteomic profile of GISTs using two-dimensional difference gel electrophoresis. The prognostic performance of biomarker candidates was examined using a large-scale sample set and specific antibodies. Results: We identified 43 protein spots whose intensity was statistically different between GISTs with good and poor prognosis. Mass spectrometric protein identification showed that the 43 spots corresponded to 25 distinct gene products. Eight of the 43 spots derived from pfetin, a potassium channel protein, and four of the eight pfetin spots had a high discriminative power between the two groups. Western blotting and real-time PCR showed that pfetin expression and tumor metastasis were inversely related. The prognostic performance of pfetin was also examined by immunohistochemistry on 210 GIST cases. The 5-year metastasis-free survival rate was 93.9% and 36.2% for patients with pfetin-positive and pfetin-negative tumors, respectively (P < 0.0001). Univariate and multivariate analyses revealed that pfetin expression was a powerful prognostic factor among the clinicopathologic variables examined, including risk classification and c-kit– or platelet-derived growth factor receptor A mutation status. Conclusions: These results establish pfetin as a powerful prognostic marker for GISTs and may provide novel therapeutic strategies to prevent metastasis of GIST.

Malignant peripheral nerve sheath tumors : an immunohistochemical study in relation to ultrastructural features

The constituent cells in malignant peripheral nerve sheath tumors were examined by studying the expression of immunohistochemical markers for Schwann cells and perineurial cells in relation to ultrastructural features in 12 malignant peripheral nerve sheath tumors. Ultrastructural studies demonstrated mixed proliferation of Schwann cells, perineurial cells, fibroblastic cells, and primitive cells in many malignant peripheral nerve sheath tumors. Expression of S-100 protein was well correlated with Schwann cell-like differentiation of tumor cells. However, Leu-7 and epithelial membrane antigen, which have been considered to be specific to Schwann cells and perineurial cells, respectively, were common to Schwann cells, perineurial cells, and primitive cells. The common immunophenotypic expression suggests a close relationship among these cell types. The unusual expression of cytokeratin could be explained by the plasticity of intermediate filament expression.

Mechanism of pain in osteoid osteomas: an immunohistochemical study.

Osteoid osteoma is a benign bone tumour characterized by pain which is relieved by non‐steroidal anti‐inflammatory drugs (NSAIDs), such as aspirin. To clarify the mechanism of the pain, five osteoid osteomas were studied immunohistochemically using polyclonal antibodies against prostaglandin E2 (PGE2), S‐100 protein and protein gene product 9.5 (PGP 9.5). In all five cases, the pain had been relieved by NSAIDs. Nerve fibres positive for S‐100 protein and PGP 9.5 were observed in the fibrous zone, especially close to the blood vessels, around the nidus in all the lesions and also within the nidus in three lesions. PGE2 immunoreactivity was variably positive in the nidus of three lesions. In one case a large number of actively proliferating osteoblasts reacted with this antibody. The other cases showed unevenly distributed PGE2 positivity which tended to be prominent in the plump osteoblasts. As control material we examined fibrous dysplasia (3 cases), osteosarcomas (3) and giant‐cell tumours of bone (3). The plump osteoblastic tumour cells of three osteosarcomas and the bone‐forming cells in two cases of fibrous dysplasia gave a positive reaction for PGE2. No S‐100 or PGP 9.5 immunoreactive nerve fibres were seen in these lesions. It is concluded that the presence of nerve fibres alone might play a more important role in mediation of pain in osteoid osteomas than some effects of osteoblast‐produced PGE2 on the nerves and proliferated blood vessels.

Epithelioid angiosarcoma of bone

Angiosarcoma of bone is a rare, high-grade sarcoma of vascular origin. This article describes an epithelioid angiosarcoma in the humerus of a 48-year-old man. A multilocular osteolytic lesion with undefined margins and destroyed cortical and medullary bone, associated with a large soft tissue mass was demonstrated radiologically in the proximal metaphysis of the right humerus. The tumor, resected by amputation, was composed mostly of proliferating malignant cells with an epithelioid morphology. It had a predominantly sheet-like growth pattern, and an occasional pseudoglandular or alveolar arrangement, mimicking an adenocarcinoma. The dilated anastomotic vascular spaces lined by epithelioid endothelial cells and the intracytoplasmic lumina/vacuoles that sometimes contained erythrocytes suggested focal endothelial differentiation. On immunohistochemical investigation, many neoplastic cells expressed cytokeratin and endothelial markers: factor-VIII related antigen, CD31, and UEA-I. The ultrastructure of the tumor was consistent with that of an angiosarcoma. Our patient died of disease shortly after the diagnosis, implying an aggressive clinical course. Awareness of the existence of skeletal epithelioid angiosarcoma, combined with the identification of intracytoplasmic lumina, or at least small vasoformative foci, and immunohistochemical positivity for endothelial markers provide the best guide for distinguishing this tumor from metastatic carcinomas.

Differentiation and proliferative activity in benign and malignant cartilage tumors of bone

To assess the histological grade in benign and malignant cartilage tumors of bone by more objective methods, we examined the differentiation and proliferative activity of tumor cells in six enchondromas, five chondroblastomas, and 13 chondrosarcomas immunohistochemically. A variable number of cells in all tumors showed S-100 protein and vimentin immunoreactivity. In fully differentiated cartilage of enchondromas and low grade chondrosarcomas, tenascin, which is an extracellular matrix glycoprotein, was present in small amounts or absent but was increased at the periphery of tumor lobules and even in the matrix throughout the high grade chondrosarcomas. Higher rate and intensity of proliferating cell nuclear antigen (PCNA) reactivity were found in chondrosarcomas, especially in spindle-shaped cells of high grade tumors, than in enchondromas. The distribution of PCNA-positive cells almost corresponded to the regions with tenascin reactivity. One tumor of high grade chondrosarcoma showed p53 protein immunoreactivity. Aberrant expression of cytokeratin was observed in four chondroblastomas. The expression of desmin was identified in relatively large proportions of enchondromas and chondrosarcomas, regardless of their benign or malignant nature and histological grade. Smooth muscle or muscle-specific actins also were present in a smaller number of tumors. Based on these findings, it is concluded that unusual staining characteristics were present, in addition to those of a chondroblastic nature, in the cartilage tumors of bone. Tenascin and PCNA positivity of various degrees in all chondroblastomas may suggest that they are chondrogenic tumors having a relatively high proliferative activity, albeit their benign clinical course. Proliferative activity of tumor cells in enchondromas and chondrosarcomas correlated well with their histological grade. Tenascin may play a role in promoting tumor cell proliferation of cartilagenous neoplasms and, on the other hand, the alterations of extracellular matrix involving tenascin synthesis seem to be a result of tumor development.

Angiomatoid (malignant) fibrous histiocytoma: A peculiar low‐grade tumor showing immunophenotypic heterogeneity and ultrastructural variations

To clarify the cellular differentiation features and facilitate diagnosis of angiomatoid (malignant) fibrous histiocytoma (AFH), four cases of AFH were examined by clinicopathologic, immunohistochemical and ultrastructural analyses. The age of the patients ranged from 10 to 24 years (mean, 17 years) and the sex distribution was equal. All cases were of subcutaneous origin: three arose in the trunk and one in the upper extremity. All patients presented with systemic symptoms, including inflammatory signs and anemia. After a mean follow up of 11 years 3 months, all patients were alive and well, although one patient twice developed local recurrence after surgery. All cases presented as multinodular, cystic and hemorrhagic tumors ranging in size from 4 to 11 cm (mean, 8 cm) and were characterized by sheets of bland spindle or round cells with oval nuclei within a circumscribed nodule often surrounded by a lymphocytic cuff. One tumor showed predominantly round cell morphology similar to Ewings sarcoma/primitive neuroectodermal tumor. All cases (100%) exhibited immunoreactivity for vimentin, desmin, CD68 and CD57 (Leu‐7). Three cases (75%) were positive for synaptophysin, and reactivity for α‐smooth muscle actin, epithelial membrane antigen, neuron‐specific enolase and CD99 (O‐13) was present in two cases (50%) each. The three cases examined by electron microscopy had a mixture of fibrohistiocytic, myofibroblastic and undifferentiated cells containing cytoplasmic processes and dense‐core granules. It is important for accurate diagnosis of this peculiar soft‐tissue tumor to recognize that it has a variety of immunophenotypes, such as histiocytic, myofibroblastic, epithelial and neural, and may occasionally have a predominantly round cell morphology.

Mechanism of pain and cytoskeletal properties in angioleiomyomas: An immunohistochemical study

Angioleiomyoma is a solitary subcutaneous tumor characterized by pain in about half of patients with this tumor, and the pathogenesis of this pain has been a cause of much debate. To clarify the mechanism of pain and cytoskeletal property of tumor cells, 50 angioleiomyomas were studied clinicopathologically and immunohistochemically. The tumors occurred preferentially on the extremities, particularly the lower leg (46%), and the female to male ratio was 1.9:1. They were classified into three histological subtypes: (i) solid (30 cases); (ii) venous (15 cases); and (iii) cavernous (five cases). The pain and/or tenderness were present in 26 out of 49 patients (52%), in which small nerve fibers immunoreactive for S‐100 protein and PGP9.5 were identified within the capsule of 20 tumors (77%) and the tumor stroma of 18 (69%), irrespective of the histological subtypes. In 24 patients where the pain was absent or unknown, nerves were observed within the capsule of 19 tumors (79%) and tumor parenchyma of 10 (42%). Many cells in all 50 tumors were positive for a‐smooth muscle actin, and a relatively large number of cells in many tumors were positive for vimentin, desmin and collagen type IV. Also, cytokeratin (CAM5.2) reactivity was scattered in a few cells of four tumors. From these findings, the peculiar pain of angioleiomyomas could be mediated by the nerve fibers especially located within the tumor parenchyma. Although the expression of intermediate filaments in angioleiomyomas was heterogeneous, the overall cytoskeletal features were of smooth muscle cell differentiation.