Shizuo Kojya

Frequent mutations of Fas gene in nasal NK/T cell lymphoma

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling through binding of Fas ligand. Mutation of Fas gene in lymphoid cells results in accumulation of these cells, which might thus contribute to lymphomagenesis. We examined the open reading frame of Fas cDNA in 14 cases of nasal NK/T-cell lymphoma. Mutations of Fas gene were detected in seven (50%) of 14 cases which comprised four frameshift, two missense, and one silent mutations. Frameshift mutations were caused by insertion of 1 bp (A) at nucleotide 1095 in two cases and by deletion of 1 bp at nucleotide 597 and at 704, respectively, in one each. Mouse T-cell lymphoma cells transfected with two missense mutated genes and frameshift mutations caused by insertion of 1 bp (A) at nucleotide 1095 were resistant to apoptosis induced by the anti-Fas antibody. These findings suggested that accumulation of lymphoid cells with Fas mutations provides a basis for the development of nasal NK/T-cell lymphoma.

Mutations of the p53 gene in nasal NK/T-cell lymphoma.

Mutations of the p53 tumor suppressor gene are reported in various kinds of malignancies including lymphomas. However, p53 gene mutations in nasal NK/T-cell lymphoma have not been reported because most parts of tumors are necrotic and a small amount of living tumor tissues is available for the molecular study. Expression and mutations of the p53 gene were examined in the paraffin-embedded specimens of the nasal lesions from 42 Chinese (Beijing and Chengdu) and Japanese (Okinawa and Osaka) patients with nasal NK/T-cell lymphoma by the immunohistochemistry and single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified products followed by direct sequencing. Thirty single-nucleotide substitution mutations were observed in 20 of 42 cases (47.6%). Among the 30 mutations, 18 were missense (mainly G:C to A:T transitions), 9 were silent, and 1 was a nonsense mutation. The remaining 2 mutations involved intron 5 and exon 5 terminal points. Abnormal expression of the p53 protein was also observed in 19 of 42 (45.2%) cases. The incidence was significantly (4-fold) higher in the cases of Osaka than those in other areas, although the incidence of p53 mutations in the cases of Osaka was one-half to one-third of those in the other three areas. The results may suggest some racial, environmental, or lifestyle differences in the cause of nasal tumorigenesis.

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.

Life‐style and environmental factors in the development of nasal NK/T‐cell lymphoma: A case–control study in East Asia

Cases of nasal NK/T‐cell lymphoma (NKTCL) occur occasionally in Asian and Latin American countries but rarely in Western countries. The etiological role of life‐style and environmental factors in nasal NKTCL was investigated. Five university hospitals in Japan and one each in Korea and China participated in this study; a total of 88 cases and 305 hospital controls were accrued during 2000–2005. The odds ratio (OR) of NKTCL obtained after adjustments of age, sex and country was 4.15 (95% confidence interval (CI), 1.74–9.87) for farmers, 2.81 (CI, 1.49–5.29) for producers of crops, 4.01 (CI, 1.99–8.09) for pesticide users, 11.65 (CI, 1.17–115.82) for residents near garbage burning plants, 2.95 (CI, 1.25–6.95) for former drinkers, and 0.49 (CI, 0.23–1.04) for current smokers. The ORs for crop producers, who minimized their exposure to pesticides by using gloves and glasses, and sprinkling downwind at the time of pesticide use, were 3.30 (95% CI, 1.28–8.54), 1.18 (95% CI, 0.11–12.13) and 2.20 (95% CI, 0.88–5.53), respectively, which were lower than those for producers who did not take these precautions. Exposure to pesticides and chemical solvents could be causative of NKTCL. Taken together, life‐style and environmental factors might be risk factors for NKTCL.

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.

Primitive neuroectodermal tumor in sinonasal region

An elderly woman having a nasal tumor diagnosed as alveolar rhabdomyosarcoma was referred to our hospital. Histological reexamination of the patient revealed that the tumor was composed of lobules of compactly arranged small round atypical cells. The atypical cells contained large oval to round vesicular nuclei, and scanty cytoplasm showing PAS positive glycogen material. The tumor cells were immunohistochemically positive for NSE, S-100 protein and vimentin, but showed negative reaction for myoglobin, desmin, EMA, keratin, LCA, chromogranin and MIC2. Ultrastructurally, the tumor cells contained a few cluster of glycogen particles and less organellae and filaments, and there were no cytoplasmic processes, neurosecretory granules or neurofilaments. Pathological findings suggested primitive neuroectodermal tumor resembling extraskeletal Ewings sarcoma, but negative reaction for MIC2 immunohistochemically could not ascertain the diagnosis. Location of the tumor and age of the patient of the present case were unusual.

Familial nasal NK/T-cell lymphoma and pesticide use

Familial occurrence of nasal NK/T‐cell lymphoma (NNKTCL) in pesticide users is presented. The proband (71 years old, male) and son (39 years old) were both diagnosed with NNKTCL within interval of 26 months. Laboratory data showed slight anemia, with no abnormal cells in peripheral blood. They and their wives were farmers and used large amounts of pesticides (fungicides and insecticides) in the hothouse. NNKTCL did not develop in the wives. Probands father was diagnosed with malignant lymphoma of the neck and died of the disease. Genetic analyses of the peripheral blood leukocytes and tumor tissues did not show p53 and k‐ras gene mutations and microsatellite instability. Metaphase cells from peripheral blood leukocytes bore specific marker chromosomes (father, 44XY,−14,−17,−18,−22,+2mar; son, 46XY,−17,+1mar). Environmental exposures to pesticides in conjunction with familial or genetic factors might increase the risk for NNKTCL. Am. J. Hematol. 66:145–147, 2001.

Adult T-cell leukemia/lymphoma with multiple integration of HTLV-1 provirus presenting as an isolated paranasal sinus tumor: A case report

Adult T‐cell leukemia/lymphoma (ATLL) is a highly aggressive T‐cell lymphoma and etiologically associated with human T‐lymphotropic virus type 1 (HTLV‐1). Patients with ATLL commonly present with leukemic changes, systemic lymphadenopathy, and/or extranodal lesion and have very poor prognosis.

Lethal Midline Granuloma in Okinawa with Special Emphasis on Polymorphic Reticulosis

Lethal midline granuloma (LMG) is a clinical term used to describe a condition which may be manifested histologically as Wegeners granulomatosis (WG), polymorphic reticulosis (PR), and malignant lymphoma (ML). WG is an inflammatory disease, and PR and ML are considered to represent a neoplastic proliferation of lymphoreticular cells. In this report, twenty‐two cases of LMG in Okinawa were examined. The frequency of LMG per 100,000 outpatients of the ear, nose and throat clinic in Okinawa was 67, and the higher frequency of PR (27) and ML (34) in Okinawa than in other districts of Japan was characteristic. Polymerase chain reaction, in situ hybridization, and immunohistochemical studies showed that the proliferating cells in PR were CD43+ and simultaneously contained Epstein‐Barr viral genome in their nuclei. The higher frequency of PR and ML in Okinawa is discussed in conjunction with a review of pertinent literature: multiple factors including genetic, viral environmental, and socioeconomic factors seem to affect the frequencies of these diseases.

Site-specific localization of Epstein-Barr virus in pharyngeal carcinomas.

In this study, the correlations of factors with Epstein‐Barr virus (EBV)‐association were investigated in 50 patients with nasopharyngeal carcinoma (NPC), 61 with oropharyngeal carcinoma (OPC), and 55 with hypopharyngeal carcinoma (HPC) in Okinawa and Osaka prefectures in Japan. The incidence of pharyngeal carcinoma in Okinawa was previously found to be higher than that in Osaka; the incidence of OPC was approximately 6 times higher and that of HPC was two times higher. The EBV genome was detected in the tumor cells of the present patients; 83% of the Okinawa and 92% of the Osaka NPC patients. The EBV genome was not detected in OPC or HPC. A univariate analysis showed that sex, the location of the tumor, histology, and the degree of lymphocytic infiltration correlated with the EBV‐positive rate. A multivariate analysis revealed that only the location of the tumor was independently correlated with the EBV‐positive rate. Histology and tumor size were factors affecting the prognosis of the patients with NPC. The NPC of poorly differentiated type frequently showed the EBV genome, and NPC with lymphocytic infiltration showed a more favorable prognosis compared to the other NPC types. These findings suggest that latent genes of EBV expressed in cancer cells might trigger a cytotoxic T cell reaction against the cancer.