Norifumi Naka

Prognostic factors in angiosarcoma : A multivariate analysis of 55 cases

Data for prognostic factors in angiosarcoma (AS) are limited, prompting a large‐scale study of AS with multivariate analysis. To analyze prognostic factors in angiosarcoma (AS), clinical and histologic findings in 55 patients collected from hospitals in Japan were reviewed. Prognostic factors were evaluated by univariate and multivariate Coxs proportional hazards models. The study involved 32 males and 23 females, ages 18–93 (median, 69) years. The primary sites of tumors included head and neck (32 cases), trunk (10), extremities (3), spleen (3), breast (3), and other (4). The overall 2‐year survival rate was 21%. Univariate analysis of clinical factors including age, sex, size and depth of tumor, tumor‐related symptoms, interval between onset of symptoms and admission, surgical procedures, adjuvant chemotherapy, and adjuvant radiotherapy showed that age, tumor size, and mode of treatment were significant for survival. Histologic factors analyzed were mitotic counts, cellularity, cellular pleomorphism, extent of necrosis, vascular differentiation, and nonspecific diagnosis. Only mitotic counts were significant for prognosis. Multivariate analysis on these four factors revealed that tumor size, mode of treatment, and mitotic counts were independent prognostic factors.

Use of immunohistochemical procedures in diagnosing angiosarcoma : Evaluation of 98 cases

Background. Differential diagnosis of angiosarcoma, predominantly showing a non‐ or poorly vasoformative proliferation from other types of sarcomas, poorly differentiated carcinomas, and amelanotic melanoma, is often problematic.

Synovial sarcoma is a stem cell malignancy.

Synovial sarcoma (SS) is a malignant soft tissue tumor characterized by its unique t(X;18)(p11;q11) chromosomal translocation leading to the formation of the SS18‐SSX fusion gene. The resulting fusion protein product is considered to play as an aberrant transcription factor and transform target cells by perturbing their gene expression program. However, the cellular origin of SS is highly debated. We herein established two novel human SS cell lines, named Yamato‐SS and Aska‐SS, and investigated their biological properties. We found the self‐renewal ability of these cells to generate sarcospheres, to form tumors in serial xenotransplantation and reconstitute the tumor phenotypes without fractionation by any surface markers. Both SS cells as well as clinical tissue specimens from 15 patients expressed the marker genes‐associated stem cell identity, Oct3/4, Nanog, and Sox2. We also found that both SS cells displayed limited differentiation potentials for mesenchymal lineages into osteocytes and chondrocytes albeit with the expression of early mesenchymal and hematopoietic lineage genes. Upon SS18‐SSX silencing with sequence‐specific siRNAs, these SS cells exhibited morphological transition from spherical growth in suspension to adherent growth in monolayer, additional expression of later mesenchymal and hematopoietic lineage genes, and broader differentiation potentials into osteocytes, chondrocytes, adipocytes, and macrophages in appropriate differentiation cocktails. Collectively, these data suggest that a human multipotent mesenchymal stem cell can serve as a cell of origin for SS and SS is a stem cell malignancy resulting from dysregulation of self‐renewal and differentiation capacities driven by SS18‐SSX fusion protein. STEM CELLS 2010;28:1119–1131

Immunohistochemical Detection of bcl-2 and p53 Proteins and Apoptosis in Soft Tissue Sarcoma: Their Correlations with Prognosis

Information on prognostic factors is essential to establish appropriate therapeutic modalities for soft tissue sarcoma (STS). To evaluate the biological nature and prognostic factors of STS, p53 and bcl-2 expression was immunohistochemically studied on paraffin-embedded sections from 70 patients with STS in the extremities and trunk. In addition, the degree of apoptosis was examined by in situ end-labeling. Histologic diagnoses in these cases were malignant fibrous histiocytoma in 29 cases, liposarcoma in 11, synovial sarcoma in 11, leiomyosarcoma in 5, malignant neurogenic tumor in 5, and others in 9. Tumor cells in 31 of 70 cases (44%) showed positive nuclear staining for p53 protein. There was no correlation between p53 expression and tumor size, histologic grade, argyrophilic nucleolar organizer region (AgNOR) count, cellularity and extent of neerosis. Expression of p53 did not correlate with survival of patients. Tumor cells in 24 of 56 cases (43%) were positive for bcl-2 protein expression. The frequency of bcl-2 expression in the tumor cells showed a direct proportion to tumor size (> or = 10 vs. < 10 cm) but inverse proportion to AgNOR counts and cellularity. The 5-year survival rate in patients with bcl-2-positive tumors (87%) was more favorable than in those with bcl-2-negative tumors (53%; p < 0.05). The frequency of apoptosis in low-grade STS was significantly higher than that in the intermediate and high-grade STS (p < 0.001). Extent of necrosis, a well-known prognostic indicator in STS, was not correlated with the frequency of apoptosis. Multivariate analysis showed that cellularity, bcl-2 and AgNOR counts were independent prognostic factors in patients with STS. The current study revealed that STS with a higher expression of bcl-2 had lower proliferative activity and larger size than those without. Immunohistochemical detection of bcl-2 is useful for predicting prognosis in patients with STS.

Mutations of p53 tumor-suppressor gene in angiosarcoma

Transgenic mice deficient for the p53 gene were reported to frequently develop angiosarcoma (AS), suggesting that alterations in the gene are associated with tumorigenesis of AS. However, little is known about genetic changes, including p53 gene alterations, in human AS because of its rarity. We analyzed p53 mutations on paraffin‐embedded specimens from 33 patients with AS by polymerase chain reaction–single‐strand conformation polymorphism (PCR‐SSCP) followed by direct sequencing. Age of patients ranged from 18 to 91 (median 70) years, with a male to female ratio of 1.5:1. Sites of tumor were the head in 13 patients, the trunk in 4, the extremities in 4, the heart in 4, bones in 2 and others in 6. PCR‐SSCP revealed aberrant mobility shifts of bands in 17 cases: 11 in exon 5, 5 in exon 7 and 4 in exon 8. Direct sequencing on these 17 cases revealed a total of 20 mutations. The frequency of p53 mutations was different by site of tumors: 7 of 13 in head, all 4 in extremities, 2 of 4 in heart and none of 4 in trunk. Our findings suggest that occurrence of p53 mutation is a major pathway for development of human AS.Int. J. Cancer 71: 952‐955, 1997.

Orphan receptor tyrosine kinase ROR2 as a potential therapeutic target for osteosarcoma

Osteosarcoma is the most prevalent bone malignant tumor in children and adolescents, and displays heterogeneous histology and high propensity for distant metastasis. Although adjuvant chemotherapy remarkably improved treatment outcome over the past few decades, prognosis for osteosarcoma patients with pulmonary metastasis is still unsatisfactory. To identify novel therapeutic targets for osteosarcoma, we investigated the gene expression profile of osteosarcomas by cDNA microarray analysis and found transactivation of receptor tyrosine kinase‐like orphan receptor 2 (ROR2) expression in the majority of osteosarcoma samples. Treatment of osteosarcoma cell lines with siRNA against ROR2 significantly inhibited cell proliferation and migration. We also identified wingless‐type MMTV integration site family, member 5B (WNT5B) as a putative ROR2 ligand and that the physiological interaction of WNT5B and ROR2 could enhance cell migration, indicating the possible roles of ROR2 and WNT5B in the metastatic property of osteosarcoma cells. Taken together, our findings revealed that the WNT5B/ROR2 signaling pathway is a promising therapeutic target for osteosarcoma. (Cancer Sci 2009; 100: 1227–1233)

Prognostic significance of activated AKT expression in soft-tissue sarcoma.

Purpose: AKT is a serine/threonine kinase which is important in tumorigenesis. Several molecules involved in AKT pathway are dysregulated in various kinds of human cancers. Patients and Methods: Ninety-three patients (53 males and 40 females), ages ranging from 19 to 77 years (median, 57 years), with localized soft-tissue sarcomas arising in the trunk and extremities, were analyzed. Immunoperoxidase procedure (avidin-biotin complex method) was done on paraffin-embedded sections with anti–phosphorylated AKT (Thr308), anti–phosphorylated p44/42 extracellular signal–regulated kinase 1 and 2 (ERK1/2) (Thr202/Tyr204), anti–phosphorylated forkhead in rhabdomyosarcoma (FKHR) (Ser256), and anti-Ki 67 antibodies. Expression levels of phosphorylated AKT (p-AKT), phosphorylated ERK1/2 (p-ERK1/2), and phosphorylated FKHR (p-FKHR) were categorized as either weaker (level 1) or equal to or stronger (level 2) compared with those in the endothelial cells of the same specimens. Percentage of cells showing intranuclear staining with Ki-67 was shown as the Ki-67 labeling index (LI). Cases were divided into two groups: level 1, Ki-67 LI < 20%; level 2, Ki-67 LI ≥ 20%. Results: Twenty-six (28.0%), 6 (6.5%), and 46 (44.1%) of the tumors showed level 2 expression for p-AKT, p-ERK1/2, and Ki-67 LI, respectively. Tumors with level 2 p-AKT expression showed a higher ratio of level 2 p-FKHR expression (P < 0.01). Multivariate analysis revealed p-AKT expression and Ki-67 LI to be independent prognosticators for overall survival, and p-AKT expression for disease-free survival. Conclusion: p-AKT expression level is a significant prognosticator in soft-tissue sarcoma.

Expression of Epstein-Barr virus latent infection genes and oncogenes in lymphoma cell lines derived from pyothorax-associated lymphoma

Malignant lymphomas frequently develop in the pleural cavity of patients with long‐standing pyothorax. The term pyothorax‐associated lymphoma (PAL) has been proposed for this type of tumor. Most PALs are diffuse lymphomas of the B‐cell type and contain Epstein‐Barr virus (EBV) DNA. We have established 2 lymphoma cell lines from biopsy specimens of PAL cases, OPL‐1 and OPL‐2, and examined their growth characteristics and the expression of EBV latent infection genes and oncogenes. OPL‐2 exhibited a more rapid growth and higher saturation density than OPL‐1, and only OPL‐2 exhibited colony‐forming activity in soft agar. OPL‐1 and ‐2 were positive for B‐cell differentiation markers and showed clonal surface immunoglobulins. Both lines contained a single predominant form of episomal EBV DNA, indicating clonal cellular proliferation of an EBV‐infected progenitor cell. OPL‐1 and ‐2 contained type B and A EBV genome, respectively. Expression of EBV nuclear antigen (EBNA)2 mRNA and protein was detected by Northern and Western blot analysis in OPL‐1, but not in OPL‐2. On the other hand, the expression of latent membrane protein (LMP) I mRNA in both OPL‐1 and ‐2 was extremely weak and detectable only by reverse transcription‐polymerase chain reaction. Protein expression of LMPI was not observed by Western blot analysis or immunocytochemistry. Both lines expressed c‐myc mRNA. Only OPL‐1 expressed mRNA of c‐fgr, an oncogene whose expression is upregulated by EBNA2. Both OPLs expressed bcl‐2 mRNA without detectable expression of LMPI protein.

Clinical implications of serum C-reactive protein levels in malignant fibrous histiocytoma.

Paraneoplastic syndromes (PNSs) associated with mesenchymal tumors are uncommon. Previous reports sporadically described inflammatory PNSs with elevated serum C‐reactive protein (CRP) levels and leukocytosis in patients with inflammatory malignant fibrous histiocytoma (MFH) of soft tissue; however, the relationship between other subtypes of MFH and PNS has not been extensively investigated. Forty‐six patients with primary MFH of soft tissues who underwent radical surgery were retrospectively analyzed. These patients were divided into 2 groups according to preoperative serum CRP level: normal (<1.0 mg/dl) and elevated (≥1.0 mg/dl). The correlation between serum CRP level and several clinicopathologic factors was analyzed. Correlation between preoperative serum CRP level and metastasis‐free and overall survival was also investigated by univariate and multivariate analyses. Elevated preoperative serum CRP levels were found in 65% of patients with a mean of 3.7 mg/dl. There were statistically significant relationships regarding tumor size, depth, histologic subtypes, grade, stage and metastatic rate among normal and elevated CRP groups (p < 0.001, p < 0.02, p < 0.005, p < 0.001, p < 0.001 and p < 0.05, respectively). When the tumor was removed, the elevated CRP level subsided into the normal range and other abnormal laboratory findings diminished in all cases. In 11/14 relapsed cases that showed elevated CRP preoperatively, the serum CRP level re‐elevated with tumor relapse. The normal CRP group showed significantly more favorable prognosis than the elevated CRP group in metastasis‐free and overall survival on univariate analysis (p < 0.02, p < 0.05, respectively). Patients with MFH frequently present with an inflammatory PNS, such as elevated serum CRP level, which can be a useful marker of disease activity and a valuable prognostic indicator.

Expression of SSX genes in human osteosarcomas

Norifumi NAKA*, Nobuhito ARAKI, Hirofumi NAKANISHI, Kazuyuki ITOH, Masayuki MANO, Shingo ISHIGURO, Diederick R.H. DE BRUIJN, Akira MYOUI, Takafumi UEDA and Hideki YOSHIKAWA Department of Orthopaedic Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Department of Pathology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Department of Human Genetics, University Medical Center, Nijmegen, the Netherlands Department of Orthopaedics, Osaka University Medical School, Osaka, Japan