Hiroyuki Kanno

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.

Large-vessel arteritis associated with chronic active Epstein-Barr virus infection

This report describes an autopsy case of large-vessel arteritis associated with chronic active Epstein-Barr virus (EBV) infection in a 10-year-old Japanese girl. All of the 3 main coronary arteries, bilateral common carotid and subclavian arteries, abdominal aorta and its major branches, and bilateral common iliac arteries were involved, and all showed aneurysmal dilation of the lumens. Histopathologic examination revealed mesoarteritis characterized by moth-eaten-appearing destruction of the medial elastic laminae, with T lymphocyte infiltration around the vasa vasorum and severe intimal thickening. The EBV DNA genome was detected in the diseased aortic tissue by polymerase chain reaction, and in the infiltrating lymphocytes by in situ hybridization. The clinical symptoms and histopathologic manifestations of the arterial lesions in this patient were obviously different from those of Kawasaki disease and Takayasu arteritis, and the arteritis was considered to be associated with the EBV infection.

Immunophenotypic and genotypic characterization of nasal lymphoma with polymorphic reticulosis morphology

Nasal lymphoma with polymorphic reticulosis (PR) morphology is now categorized as T/natural killer (T/NK) cell lymphoma. In this study, immunophenotypes and genotypes of proliferating cells in 21 cases with PR were examined. The patients included 13 men and 8 women ranging in age from 20 to 74 (median 37) years. All patients presented with lesions in the upper respiratory tract, mostly in the nasal cavity. Histological specimens obtained from the primary lesions (19 cases) and metastatic cervical lymph nodes (2 cases) were used for analyses. Histologically, polymorphous proliferation was found in 20 cases, and these were thus diagnosed as PR. A monomorphous pattern was found in the remaining last case. Immunohistochemical analysis revealed that the proliferating cells were CD56 (123C3)+ and/or CD16 (2H7)+, TIA‐1+ and frequently stained CD3 ϵ+. Tumor cells were frequently stained positively with monoclonal antibodies (mAbs) for T lymphocytes, but were negative for T‐cell receptor (TCR) β and δ chain expression. In situ hybridization analysis using an Epstein‐Barr virus‐encoded early RNA 1 (EBER‐1) probe revealed positive signals in 13 of the 15 cases examined. Southern blotting analysis for clonality of the Epstein‐Barr virus (EBV) genome in 12 positive cases confirmed the presence of monoclonal proliferation in 7 cases. The pattern of TCR γ chain gene rearrangement was examined by PCR analysis of DNA from tumor tissues by the denaturing gradient gel electrophoresis method. The results demonstrated no clonal rearrangement in any of the 21 cases examined, including 7 cases with proven clonal proliferation of EBV‐infected cells, indicating the absence of T‐cell clones. Our findings strongly suggested that nasal T‐cell lymphoma is in fact a NK cell lymphoma. Int. J. Cancer 81:865–870, 1999.

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.

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H2O2 but not O·-2 produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H2O2 as ROS stress thereafter. The H2O2-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H+-monocarbohydrate transporter, increased the H2O2-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.

Angiogenesis in Malignant Fibrous Histiocytoma

The significance of neovascularization for tumor growth and metastasis has recently been postulated for human cancers; increased microvessel density correlates with increased frequency of metastasis. In the present study, microvessel density was examined in 42 cases of malignant fibrous histiocytoma (MFH). Microvessels were defined as lumens surrounded by anti-factor-VIII-related antigen (FVIII-RA)-antibody-stained endothelium, and counted in a x 400 field. The number of microvessels varied from 4 to 79 (median 14.5). When cases were divided into groups with less than or greater than 20 microvessels, there were no prominent differences in age distribution, sex ratio, size of tumor, depth of tumor, and histologic subtypes between the two groups. The number of microvessels in 19 cases with and 22 cases without metastasis was 19.4 +/- 14.9 and 19.6 +/- 17.4, respectively. Angiogenesis is apparently not a key factor in the formation of metastasis by MFH.

Low frequency of HLA-A*0201 allele in patients with Epstein-Barr virus-positive nasal lymphomas with polymorphic reticulosis morphology

Lymphoproliferative diseases of the nasal cavity and paranasal sinuses occur frequently in Asian countries and are histologically categorized as monomorphic ordinary lymphoma and polymorphic reticulosis (PR) with apparent inflammatory cell infiltration. The large atypical cells in PR show natural‐killer cell nature and frequently contain Epstein‐Barr virus (EBV) DNA. Among the EBV genes involved in latent infection, those encoding EBV latent membrane proteins are frequently expressed in PR. Several cytotoxic T‐lymphocyte (CTL) defined epitopes have been mapped to latent membrane proteins restricted with HLA‐A2, ‐A11 or ‐A24 antigens. Thus, the HLA‐A allele may affect the development of PR. To examine this possibility, HLA‐A alleles of 25 patients with EBV+ PR were determined with low‐resolution polymerase chain reaction‐based typing using HLA‐A locus sequence‐specific primer combinations. The frequency of HLA‐A alleles including HLA‐A2 and ‐A24 antigens in PR patients was lower than that in the normal Japanese population, but the difference was not significant. Since HLA‐A2‐restricted CTL responses are well delineated at the A2‐subtype level, the A2‐subtype of PR cases with HLA‐A2 antigen was further determined by high‐resolution genetic typing. The frequency of HLA‐A*0201 in PR was significantly lower than in the normal population (p=0.0314). The HLA‐A*0201‐restricted CTL responses may thus function in vivo to suppress the development of overt lymphoma. Int. J. Cancer 87:195–199, 2000.

Microsatellite instability and k-ras, p53 mutations in thyroid lymphoma.

Patho‐epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B‐cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B‐cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k‐ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH (P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k‐ras gene. The k‐ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k‐ras mutation, indicating a close association between RER and k‐ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.

HLA‐A alleles of patients with pyothorax‐associated lymphoma: Anti‐Epstein‐Barr virus (EBV) host immune responses during the development of EBV latent antigen‐positive lymphomas

Pyothorax‐associated lymphoma (PAL) is an Epstein‐Barr virus (EBV) latent antigen‐positive lymphoma resembling EBV‐transformed lymphoblastoid cell lines (LCLs) and develops in non‐immunocompromised patients. Thus, deficient anti‐viral‐antigen immune responses might be involved in the development of PAL. As MHC class I–restricted cytotoxic T lymphocytes (CTLs) are the major constituent of anti‐viral immune responses, the HLA allele type and its expression may affect the development of PAL. Flow‐cytometric analyses of PAL cell lines and LCLs using the W6/32 monoclonal antibody revealed that expression of HLA class I varied among cell lines. Although one PAL cell line, OPL‐2, exhibited low expression, an LCL and another PAL cell line, OPL‐1, strongly expressed HLA class I. Among the EBV latent infection genes, EBV nuclear antigens 2, 3, 4 and 6 and latent membrane proteins can induce efficient CTL responses in combination with HLA‐A2 or ‐A11. HLA‐A alleles of PAL patients were determined using low‐resolution PCR‐based typing with HLA‐A locus sequence‐specific primer combinations. The antigen frequencies of HLA‐A2 and ‐A11 in PAL patients were not significantly different from those in the normal Japanese population. Although HLA class I antigen should be expressed during the course of lymphomagenesis, no HLA‐A alleles influenced the development of overt PALs. Int. J. Cancer 82:630–634, 1999.