Tadashi Hongyo

Nasal NK/T-cell lymphoma: epidemiology and pathogenesis

Nasal NK/T-cell lymphoma (NKTCL) is an uncommon disease, but usually shows a highly aggressive clinical course. The disease is much more frequent in Asian and Latin American countries than in Western countries, and is universally associated with Epstein–Barr virus (EBV) infection. Analyses of gene mutations, especially p53 and c-KIT, revealed the different frequencies by district. Epidemiological studies revealed the changes of the disease frequency in Korea during the period from 1977–1989 to 1990–1996. Case-control study showed that the exposure to pesticides and chemical solvents could be causative of NKTCL. Further studies including HLA antigen typing of patients is necessary to further clarify the disease mechanism.

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.

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.

Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population

We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV‐positive than non‐oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV‐positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus‐unrelated HNSCC and virus‐related HNSCC consisting of nasopharyngeal and HPV‐positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus‐unrelated HNSCC. A disruptive mutation was never found in virus‐related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus‐unrelated HNSCC. Moreover, in virus‐unrelated HNSCC, G:C to T:A transversions were more frequent in ever‐smokers than in never‐smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever‐smokers than in never‐smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus‐related and virus‐unrelated subgroups. In virus‐related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus‐unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.

Analysis of p53, K‐ras, c‐kit, and β‐catenin gene mutations in sinonasal NK/T cell lymphoma in northeast district of China

Recently we reported the different frequenties of p53 and c‐kit gene mutations among sinonasal NK/T cell lymphoma (NKTCL) in Korea, north China (Beijing), and Japan, suggesting some racial, environmental, or life‐style differences as a possible cause of nasal tumorigenesis. In this study, gene mutations in p53, c‐kit, K‐ras, and β‐catenin gene were analyzed by polymerase chain reaction (PCR)‐single strand conformation polymorphism (SSCP) followed by direct sequencing in 20 cases of sinonasal NKTCL from northeast China (Shen Yang). Age of patients ranged from 5 to 63 (median, 40.0) years. p53 gene mutations were found in eight of 20 cases (40%), with exon 4 involvement in 10% of cases. The majority was missense mutations and G:C to A:T transition was predominant. The frequency of the c‐kit and K‐ras gene mutations was low (5%), while that of the β‐catenin gene was six of 20 cases (30%). From these findings, it is concluded that nasal NKTCL in northeast China shared common features with that in Korea in the younger onset of disease compared to that in Japan and lower frequency of p53 gene mutations with infrequent exon 4 involvement compared to that in Japan and north China. These differences might be caused by migration of susceptible populations or some environmental confounding factors. (Cancer Sci 2003; 94: 297–301)

Gene mutations in lymphoproliferative disorders of T and NK/T cell phenotypes developing in renal transplant patients.

Post-transplantation lymphoproliferative disorder (PT-LPD) is characterized by lymphoid proliferation after organ or bone marrow transplantation. In Western countries, most cases of PT-LPD are B-cell–derived and Epstein-Barr virus–associated, in which alterations of c-myc, p53, and N-ras genes might play a role in disease progression. In Japan, PT-LPD of T- and NK/T-cell types are not uncommon in renal transplant patients. Mutations of p53 (exons 4 through 8), K-ras (exons 1 and 2), c-kit (exons 11 and 17), and β-catenin genes (exon 3) in 12 cases of these diseases were analyzed by PCR single strand conformation polymorphism and then by direct sequencing. p53 gene mutations were detected in 5 of 5 cases of peripheral T-cell lymphoma, 3 (60%) of 5 cases of adult T-cell leukemia/lymphoma, and 1 of 2 cases of NK/T cell lymphoma. Twenty-five percent of T and NK/T cell lymphomas showed K-ras mutations. Mutations of c-kit and β-catenin genes were found in 33% of cases. Among a total of 42 substitution mutations, 40 were transitions with involvement of CpG sites in 20 to 30% of cases. Most cases had at least one mutation that changed an amino acid, which might have provided the selection pressure for expansion. These findings suggested that p53 gene mutations might play a central role in development of peripheral T-cell lymphoma including adult T-cell leukemia/lymphoma in renal transplant patients.

Mutations of p53, c-kit, K-ras, and β-Catenin Gene in Non-Hodgkin's Lymphoma of Adrenal Gland

Malignant lymphoma of the adrenal gland is a rare disease, usually with diffuse large cell morphology and B‐cell immunophenotype, and often associated with Epstein‐Barr virus infection. In this study, mutations of p53, c‐kit, K‐ras, and β‐catenin gene were analyzed in 17 cases (13 males and four females with ages ranging from 25 to 84 years) of such lymphomas by polymerase chain reaction‐single strand conformation polymorphism followed by direct sequencing. Selected exons in each gene, representing hot spots, were analyzed. All 44 mutations detected were single‐nucleotide substitutions and 33 were missense mutations. Nineteen mutations were detected in exon 5 and/or 7 of the p53 gene in nine of 17 cases (52.9%) and 21 in exon 11 and/or 17 of the c‐kit gene in 10 of 14 cases (71.4%). Bilateral adrenal lesions in one case who had not received any adjuvant therapy showed different mutational patterns of the p53 and c‐kit genes, suggesting different clonal evolution of lymphoma between the left and right sides. Mutation at codon 13 of the K‐ras gene was detected in one of 14 cases (7.1%), and in exon 3 of the β‐catenin gene in three of 12 cases (25%). All but one mutation were transition mutations, indicating that some endogenous mutagens act in lymphomagenesis in the adrenal gland. Our results suggest that p53 and c‐kit gene mutations might play a role in adrenal lymphomagenesis.

Rapid Growth and Spontaneous Metastasis of Human Germinal Tumors Ectopically Transplanted into Scid (Severe Combined Immunodeficiency) and Scid‐nudestreaker Mice

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F2 hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST‐2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c‐nu/nu and CDl‐nu/nu). The YST‐2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude‐streaker (AKR/J‐nustr/nustr) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one‐third (36.4%) of scid mice and very rapidly in all of scid‐nu (scid/scid; nustr/nustr) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid‐nustr mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.

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.

Morphology and function of human benign tumors and normal thyroid tissues maintained in severe combined immunodeficient mice

In the improved SCID (severe combined immunodeficient) mice, various human benign tumors of the head and neck region were well maintained morphologically and functionally for 3 years until the experiments were terminated, e.g. transplanted parathyroid adenoma secreted parathyroid hormone (PTH) in the SCID mice for more than 1 year. Normal human thyroid tissue was also well maintained in the SCID mice for 3 years. Rapid and high uptake of radioiodine into the transplanted human thyroid tissue was observed. Furthermore, transplanted human thyroid tissue secreted thyroid hormone (T3) and T3 secretion was stimulated by the injection of human thyroid stimulating hormone (TSH). These findings suggest that the improved SCID mice will provide an invaluable experimental system for investigating the function of normal human tissues and the influence of endogenous and exogenous factors on human tissues.