Seiji Haraoka

CD10 and Bcl10 expression in diffuse large B‐cell lymphoma: CD10 is a marker of improved prognosis

CD10 and Bcl10 expression in diffuse large B‐cell lymphoma: CD10 is a marker of improved prognosis

Amplification and expression of a decoy receptor for fas ligand (DcR3) in virus (EBV or HTLV-I) associated lymphomas.

The recently identified decoy receptor 3 (DcR3) binds to FasL and inhibits FasL-induced apoptosis, and is considered to play a role in the immune escape system of neoplastic cells. To examine the involvement of DcR3 in the immune evasions of virus-associated lymphoma, we analyzed the amplification and expression of DcR3, using dot blot and in situ hybridization (ISH), in 45 cases, which included 17 cases with Epstein-Barr virus (EBV)-associated lymphoma (seven pyothorax-associated B-cell lymphomas (PAL); ten natural killer lymphoma (NKL)), seven cases with adult T-cell leukemia lymphoma (ATLL), 13 Hodgkins disease (eight EBV-associated cases; five non-EBV-associated cases), and eight control cases (three reactive lymphadenopathy; five non-EBV-associated-B-cell lymphoma). EBV-associated PAL and NKL exhibited DcR3 amplification and expression in lymphoma cells. ATLL also showed DcR3 expression and amplification. The cases with DcR3 amplification showed DcR3 expression; however, the expression was confined in the neoplastic cells, but not in the reactive cells. In Hodgkins disease (HD), DcR3 was expressed only in Hodgkin and Reed-Sternberg giant (H-RS) cells. However, DcR3 was not expressed or amplified in reactive lymphadenopathy. Non-EBV-associated B-cell lymphoma also rarely expressed DcR3, and showed no amplification except in two cases, in which rare expression was present. Our results suggest that EBV and HTLV-I probably use DcR3 to escape from the immune system during lymphomagenesis, or virus-infected lymphoma cells with DcR3 expression might be selected in the multistep tumorigenesis.

Mutation analysis of mitotic checkpoint genes (hBUB1 and hBUBR1) and microsatellite instability in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a neoplasm of T-lymphocytes, and human T-cell lymphotropic virus type-I (HTLV-I) is etiologically considered as the causative virus of ATLL. The karyotypes of ATLL are very complex in both number and structure, although no specific karyotype abnormalities have been identified. HTLV-I is thought to integrate its provirus into random sites in host chromosomal DNA and induces chromosomal instability. The BUB gene is a component of the mitotic checkpoint in budding yeast. Recently, human homologues of the BUB were identified and mutant alleles of hBUB1 and hBUBR1 were detected in two colorectal tumor cell lines, which showed microsatellite instability (MIN). In vitro, BUB proteins form a complex of monomers. These proteins interact with the human MAD1 gene product, a target of the HTLV-1 tax oncogene. We examined the role of checkpoint gene in the chromosomal abnormalities of ATLL by investigating mutations of hBUB1 and hBUBR1, and MIN of replication errors of BAX, insulin-like growth factor, and transforming growth factor beta type II. We analyzed ten cases with ATLL and eight B-cell lymphomas (five diffuse large cell lymphomas, three follicular lymphomas). Complex chromosomal abnormalities were detected in ATLL, while B-cell lymphomas showed only simple or minimal chromosomal abnormalities. Significant mutations/deletion of hBUB1 or hBUBR1 were detected in four of ten cases with ATLL, including two heterozygous point mutations, one homozygous point mutation, and one with a 47 bp deletion. In contrast, only one of eight B-cell lymphomas showed nonsense mutation of hBUBR1. None of the ATLL and B-cell lymphomas showed MIN. In the multistage process of leukemogenesis of ATLL, our findings indicate that mutations of mitotic checkpoint genes may play an important role in the induction of complex chromosomal abnormalities.

Intractable ulcerative colitis caused by cytomegalovirus infection: a prospective study on prevalence, diagnosis, and treatment.

PURPOSE Cytomegalovirus infection has been known to complicate ulcerative colitis. This study was designed to elucidate the prevalence and clinical features of ulcerative colitis that might point efficiently to the diagnosis of complicating cytomegalovirus infection in cases of ulcerative colitis. METHODS The study included 47 consecutive patients diagnosed to have moderate-to-severe ulcerative colitis who were treated on an inpatient basis at our department during a two-year period. A prospective examination for cytomegalovirus antigenemia was conducted in all patients with moderate-to-severe ulcerative colitis to determine the prevalence of cytomegalovirus infection among these patients. Then, the characteristic clinical and endoscopic features of ulcerative colitis complicated by cytomegalovirus infection were investigated by comparison of the cytomegalovirus-infected group with the non-cytomegalovirus-infected group. The therapeutic effects of antiviral drugs also were assessed. RESULTS Cytomegalovirus infection was detected in 16 of 47 patients (34 percent). Proportion of female patients, age at the time of determination, and proportion of patients showing corticosteroid resistance was significantly higher in the cytomegalovirus-infected group (59.1 percent) than in the non-cytomegalovirus-infected group (13.6 percent). The prevalence of endoscopically severe ulcerative colitis was higher in patients with cytomegalovirus antigenemia than in those without cytomegalovirus antigenemia (P = 0.016). Ganciclovir was administered to 12 of 16 ulcerative colitis patients with complicating cytomegalovirus infection, and was found to be effective in 8 (66.7 percent). CONCLUSIONS It is not easy to make a diagnosis of cytomegalovirus infection complicating ulcerative colitis based on clinical features, including endoscopic biopsy. On the other hand, blood examination for the detection of cytomegalovirus antigenemia in corticosteroid-resistant patients, particularly in relatively elderly patients, may enable diagnosis of cytomegalovirus infection with a high sensitivity and allow effective treatment to be administered in these patients.

Gastrointestinal T Cell Lymphoma: Predominant Cytotoxic Phenotypes, Including Alpha/Beta, Gamma/Delta T Cell and Natural Killer Cells

Gastrointestinal T cell lymphoma (TCL) is a rare subset of peripheral TCL, presenting with or without cytotoxic phenotype, a history of coeliac disease (CD) and enteropathy. However, CD is rare in Japan. Here, we describe the clinicopathological features of 18 Japanese cases. Lesions were found in the small intestine (n=13), stomach (n=3) and colon (n=2). Seven patients presented with enteropathy but none had a history of CD. Lymphomas appeared as ulceration (n=ll), tumour formation (n=6), or polypoid growth (n=1). Histologically (REAL classification), neoplastic lesions were composed of intestinal type T cell lymphoma (ITCL, n=13, including one case with NK type), anaplastic large cell (ALCL, n=2), adult T cell leukaemia/lymphoma (ATLL, n=2), and lymphoblastic type (n=1). Epstein Barr virus infection was detected by EBER-1 in situ hybridization in 6 of 11 cases with ITCL but not in the other types. ALCL expressed CD30. CD56 was expressed in 3 of 11 cases of ITCL but not in other types. Among the 10 examined cases, 8 were αβ T cell type [CD2+, CD3+, T cell receptor (TCR)δ-1-, βF1+], one was γδ T cell type [CD2+, CD3+, TCR8-1+, (βF1-], and the remaining case expressed natural killer (NK) cell type [CD2+, CD3-, CD56+, TCRδ-1-, βF1-]. Among the 8 examined cases, 3 expressed CD103 molecule, which was associated with extrathymic T cells of intraepithelial lymphocytes. All cases except ATLL expressed the cytotoxic-ity-associated molecule of TIA-1, and 11 of 14 TIA-1 positive cases expressed activated cytotoxic molecules of perforin, granzyme B, and/or Fas ligand. Despite the morphological, genetic and phenotypic heterogeneity, prognosis was poor, and 11 of 13 patients with small intestinal lesions died albeit appropriate treatment, but 3 of 4 patients with gastric or colonic lesions were still alive. The main cause of death was intestinal perforation. The latter might be due to the site specificity of small intestine and tumour cytotoxicity.

Expression of human tumor-associated antigen RCAS1 in Reed-Sternberg cells in association with Epstein-Barr virus infection: a potential mechanism of immune evasion.

RCAS1 (receptor‐binding cancer antigen expressed on SiSo cells) is present in neoplastic cells, induces apoptosis of natural killer (NK)/T cells and plays a role in immune evasion. Fas ligand (FasL) is considered to have similar roles. The Epstein‐Barr virus (EBV)–encoded latent membrane protein is expressed by malignant Hodgkin and Reed‐Sternberg (H&RS) cells of EBV‐associated Hodgkins disease (HD) and considered to be a target of cytotoxic T lymphocytes (CTLs). However, CTL response is inadequate in HD. To determine whether RCAS1 and FasL are expressed in EBV‐associated HD and participate in immune evasion, tissues of 20 EBV– and 15 EBV+ HD cases were immunohistochemically stained for RCAS1, FasL and HLA classes I and II, whose deficiencies could explain CTL escape. Lymphocytes surrounding H&RS cells tended to be CD4+ cells and rarely CD8+, TIA‐1+ (cytotoxic marker) or NK cells. HLA class I and/or II were expressed in all EBV+ HD cases, and RCAS1‐expressing H&RS cells were found in 14/15 (93%) EBV+ HD cases but only 8/20 (40%) EBV– HD cases (p < 0.05). FasL was detected in 9/15 (60%) and 7/20 (35%) EBV+ and EBV– HD cases, respectively. ssDNA‐positive (apoptotic) lymphocytes, surrounding H&RS cells, were rarely seen but were present in RCAS1+ cases (20/22 cases, 91%) rather than negative cases (0/13 cases, 0%) (p < 0.005). Our findings suggest that EBV+ H&RS cells might evade the host immune response by expressing RCAS1 rather than FasL.

Nodal T‐cell lymphoma in an HTLV‐I‐endemic area: proviral HTLV‐I DNA, histological classification and clinical evaluation

Adult T‐cell leukaemia/lymphoma (ATLL) is a human malignancy associated with human T‐cell leukaemia virus type I (HTLV‐I). The histology usually indicates a pleomorphic type, but is not consistent. To clarify the relationship between the histological classification and prognosis in ATLL, and to confirm the significance of clonal HTLV‐I integration, we reclassified 572 cases with nodal T‐cell lymphoma in which the T‐cell phenotype and/or genotype was confirmed. In all cases the clonal integration of HTLV‐I proviral DNA in the lymph nodes was examined by Southern blot analysis. In addition, anti‐ATL antigen (ATLA) determination in the serum or PCR analysis of HTLV‐I pX amplification in lymph nodes was also performed. 66/313 (21%) cases with ATLA had no evidence of clonal HTLV‐I integration. 572 cases were classified into three groups: (A) cases with clonal integration (247 cases), (B) cases with ATLA without clonal integration of HTLV‐I proviral DNA (66 cases), (C) cases without ATLA (259 cases). Histologically, groups B and C frequently demonstrated large cell type and angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD) type; however, group A tended to show a pleomorphic type. Clinically, group A showed a poorer prognosis than groups B and C.

Genetic analysis of sorted Hodgkin and Reed‐Sternberg cells using comparative genomic hybridization

Hodgkin and Reed‐Sternberg (H and RS) cells are generally considered to be the neoplastic cells of Hodgkins disease (HD); however, such cells are found only in a minority of HD lesions. Very few data have so far been published on the cyogenetic abnormalities in HD. An analysis of unknown genetic aberrations has only recently become possible through the use of comparative genomic hybridization (CGH), which is based on the competitive binding of tumor and control DNA to metaphase chromosomes. In order to exclude the reaction of non‐tumor cells, we used 100 sorted H‐RS cells as the tumor DNA, then 100 sorted reactive T cells or B cells as the control DNA. We obtained the amplified DNA, using degenerate oligonucleotide‐primed polymerase chain reaction (DOP‐PCR). In addition, to confirm whether or not the lymphocytes in the background were reactive, we performed CGH with 100 sorted B cells and 100 sorted T cells. CGH was thus performed on 9 HDs, including 6 cases of mixed‐cellularity (MC) sub‐type and 3 cases of nodular‐sclerosis (NS) sub‐type. CGH of the B and T cells showed no genetic changes in any cases. In contrast, CGH of H‐RS cells revealed both gains and losses of DNA in all 9 cases, and multiple changes were also observed. In situ hybridization showed an Epstein‐Barr‐virus infection in 5 cases of MC; however, no definite relationship was observed between the EBV infection and genetic changes. The most commonly observed genetic aberrations were a loss on 16q11/21 in 6 cases, a gain on 1p13 in 5 cases, and a gain on 7q35/36 in 5 cases. The large number of chromosomal alterations in HD suggests, therefore, that an increased degree of genetic instability play a role in the formation of H‐RS cells. Int. J. Cancer 82:250–255, 1999.

Analysis of the immunoglobulin heavy chain gene variable region of intravascular large B-cell lymphoma

Abstract Intravascular large B-cell lymphoma (IVLBL) is a rare neoplasm characterized by proliferation of lymphoma cells within the blood vessels. The cell origin of IVLBL has not yet been determined. We examined cell lineage, with immunohistochemical staining and molecular analysis, using polymerase chain reaction (PCR) of the variable region of the immunoglobulin heavy chain (Ig-VH) gene. We also investigated the cell origin using direct sequence analysis of the complementary-determining region 2 (CDR2) and framework region 3 (FR3) in six cases, consisting of two male and four female patients. The sequences in five cases showed frequent mutations. The percent homology to their closest germline genes ranged from 74.7 to 91.8%. However, one case showed a percent homology of 99.4% in CDR2 and FR3 of Ig-VH. All cases showed rearrangements of VH3 family genes. Interestingly, three of the IVLBL cases with hypermutated IgH genes showed the expression of CD5. Therefore, expression of CD5 in lymphoma cells does not indicate that the origin of IVLBL is the same as mantle cell lymphoma having the character of CD5 expression, which develops in pre-germinal center cells. Our results indicate that most IVLBLs may originate in the post-germinal center cells, based on the presence of somatic mutation in VH genes, although some heterogeneous cases are intermingled within IVLBL.

Cytoplasmic Cytokines in Lymphoproliferative Disorders: Multiple Cytokine Production in Angioimmunoblastic Lymphadenopathy with Dysproteinemia

Cytokines play an important role in the pathogenesis of lymphomas via autocrine or paracrine mechanisms, or both. Here we determined the proportion of CD3-positive T lymphocytes containing various types of cytokines in enlarged lymph nodes. Lymph nodes were obtained from 16 patients with various lymphoproliferative disorders, including 3 cases with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), 3 cases with adult T cell leuke-mia/lymphoma (ATLL), 2 cases with T-cell nonspecific malignant lymphoma (T-ML), 3 cases with B-cell diffuse large malignant lymphoma (BDL), 3 cases with histiocytic necrotizing lymphadenitis (HNL), and 2 cases with non-specific lymphadenitis (NSL). The percentages of T lymphocytes positive for cytoplasmic cytokines IL-2, IL-4, IL-5, IL-6, IL-13, TNF-α, and INF-γ were determined. The percentage of INF-γ positive T lymphocytes was high in reactive lymphadenopathy of NSL and HNL. AILD showed a high proportion of TNF-α positive T-lymphocytes, and in addition, the percentages of IL-2, IL-4, IL-5, IL-6, IL-13 and INF-γ positive T-lymphocytes were relatively higher than in other diseases. Our results supported the state of multiple hypercytokinemia typically seen in AILD and suggested that the source of the cytokines is the lymph nodes. Our results also suggested that multiple cytokine networks play an important role in the clinical and histopathological features of AILD. Modulation of the cytokine network may be the logical objective in future therapeutic strategies designed for AILD.