Midori Sugihara

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.

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.

CLINICOPATHOLOGICAL STUDY OF SEVERE CHRONIC ACTIVE EPSTEIN-BARR VIRUS INFECTION THAT DEVELOPED IN ASSOCIATION WITH LYMPHOPROLIFERATIVE DISORDER AND/OR HEMOPHAGOCYTIC SYNDROME

Chronlc active Epstein‐Barr virus (CAEBV) infection has been prevlously reported to be sometimes associated with an aggresslve clinical course. However, the role of EBV in the CAEBV Is not well clarifled. A retrospective study was performed on nine adult and five child patients (eight males and six females). Histologlcally, at first admission, the presence of neoplastic lesions could not be confirmed. The lymph nodes In half of all cases revealed paracortical hyperplasla with transformed lymphocytes (hyperplastic type). Half of the cases showed nonsuppuratlve necrosis and an Increased number of histiocytes with phagocytosis (histiocytic type). Activated histiocytes with lymphokine positivity were frequently detected in the histiocytic type. In the phenotypical study, 10 of the examined 11 cases showed Increased numbers of natural killer (NK) cells and/or CD8 positive T lymphocytes. In situ hybridization (ISH) showed EBV‐Infected lymphoid cells, but the number of EBV‐infected cells varied. Double‐labeling irnmunochemistryASH demonstrated EBV‐infected T cells, including NK cells, but not B cells. In addition, three cases showed a monoclonal dissemination of EBV terminal repetitive sequence (TR), and two cases showed oligoclonal dissemination. From those findings, monoclonal, oligoclonal and polyclonal populations of EBV‐infected T or NK cells were considered to be present in CAEBV states. During the clinical course, 12 of the 14 cases died within 5 years. Six cases died from EBV‐associated hematopoietic tumors (histiocytlc tumor, T cell lymphoma, B cell lymphoma, plasmacytoma, and NK cell leukemia); one from non‐EBV‐associated acute myeloge‐nous leukemia, and five due to hemophagocytic syndrome. The examined EBV‐associated hematopoietic tumors showed monoclonal EBV terminal repetitive sequences. There is a possibility that the monoclonal dissemination of EBV‐infected cells develops from oligoclonal or polyclonal EBV‐infected cells. And active histlocytes with lymphokine positivity were frequently detected In the cases with histologically histiocytic type. These findings seem to be related with the causes of death due to hemophagocytic syndrome.

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.

Clinical, immunohistochemical and phenotypic features of aggressive nodal cytotoxic lymphomas, including α/β, γ/δ T-cell and natural killer cell types

Abstract Cytotoxic cells include natural killer (NK) cells and cytotoxic αβ and γδ T lymphocytes (CTLs). These cells express cytotoxic molecules of T-cell restricted intracellular antigen(TIA-1), and activated cytotoxic molecules of perforin, granzyme B, and FasL. Recent studies suggest that most extranodal T-cell lymphomas are derived from CTLs, and that NK cell lymphomas are extranodal. However, only a few nodal NK and cytotoxic lymphomas have been described so far. We present here the clinicopathological features of seven cases of nodal cytotoxic T and NK cell lymphomas. The study excluded anaplastic large-cell lymphomas expressing cytotoxic molecules. The neoplastic cells of all cases contained activated cytotoxic molecules of TIA-1, granzyme B, Fas ligand, and/or perforin. Phenotypically and genotypically, four cases showed αβ T cell type [CD2+, CD3+, T-cell receptor (TCR) δ-1–, βF1+, and TCR gene rearrangement], two cases showed γδ T-cell type [CD2+, CD3+, T-cell receptor (TCR)δ–1+, βF1–, and TCR gene rearrangement], and one case showed NK cell type [CD2+, CD3-, CD56+, T-cell receptor (TCR)δ-1–, βF1–, and TCR gene germline]. Using Southern blot analysis, Epstein-Barr virus (EBV) sequences were detected in six cases, and monoclonal terminal repeat proliferation was confirmed. In addition, in situ hybridization (ISH) studies for EBV showed EBV infection in almost all neoplastic cells. Clinically, all patients presented with peripheral lymphadenopathy in high clinical stages and showed an aggressive course. Hepatosplenomegaly was detected in six cases. During the course of the disease, bone marrow and extranodal invasion were noted in five cases. The nodal type showed an aggressive clinical course in all cases but one, as did the extranodal type. The nodal type varied in phenotype, but was closely associated with EBV infection.

Basic Fibroblast Growth Factor and Fibrosis in Hodgkin's Disease

Hodgkins disease (HD) is characterized by the presence of Hodgkin and Reed-Sternberg (H-RS) cells against a hyperplastic background of reactive cells such as lymphocytes, histiocytes, plasma cells, eosinophils, neutrophils and stromal cells. In addition, the HD nodular sclerosis (NS) subtype shows characteristic fibrous bundles, while the other subtypes do not. The fibrosis is considered to correlate with multiple cytokines and cytokine networks. Basic fibroblast growth factor (bFGF), one of the potent stimulators of fibroblasts, has also been linked to the fibroproliferative process. To investigate the relationship of fibrosis and bFGF, we thus performed both immunostaining, in situ hybridization (ISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) on 25 cases of HD, which included 12 cases with NS subtype, 10 cases with mixed cellularity (MC), and 3 cases with lymphocyte predominance (LP). In NS, the expression of bFGF was stronger than that in LP and MC. In addition, the H-RS cells in NS frequently expressed bFGF. The stromal cells and histiocytes in the background expressed bFGF in NS. However, in MC and LP the number of bFGF-expressed H-RS cells was small, and the bFGF expression of background cells was rarely detected. However, the amount of bFGF varied in each case with HD NS. The above results support the possibilities that H-RS cells and background cells are a cellular source of bFGF and that the bFGF expression of those cells is also one of the influencing factors in the development of fibrosis in the HD NS subtype.

Clinicopathological findings of virus‐associated hemophagocytic syndrome in bone marrow: Association with Epstein‐Barr virus and apoptosis

Non‐neoplastic hemophagocytic syndrome (HPS), also called virus‐associated hemophagocytic syndrome (VAHS), has been thought to be a distinct clinical entity. A spontaneous recovery is common, but the prognosis of Epstein‐Barr virus (EBV)‐associated VAHS is poor. However, the role of EBV has yet to be clearly elucidated. A retrospective study of the bone marrow of 30 cases, in which the diagnosis of non‐neoplastic VAHS was clinicopathologically confirmed, was performed. We were unable to histologically confirm the presence of neoplastic lesions, especially lymphoma cell infiltration. Ten of the patients were children (aged less than 15 years) and young adults (aged under 20 years; median age, 10 years). Twenty patients were adults (aged over 21 years; median age, 48 years). Twelve of these patients died, while 18 showed a spontaneous recovery. We performed immunological staining and in situ hybridization (ISH) for EBV. To clarify the presence of apoptosis, an in situ apoptosis detection (tunnel) method was used. In situ hydridization showed an EBV‐presence in 16 of the 30 patients. In addition, the EBV‐presence was confined in the lymphocytes, especially T lymphocytes in double stainings. The number of EBV‐infected cells varied; however, the EBV presence was associated with ages. Nine of the 10 children and young adults showed an EBV‐presence, while EBV was detected in seven of the 20 adults. Especially in 10 patients aged over 49 years, no EBV was detected. According to the in situ apoptosis detection, apoptotic cells were increased in number and considered to be lymphoid cells, but not myeloid or histiocytic cells. Some apoptotic cells were phagocyted with histiocytes. Histologically, apoptosis may be one of the factors that induced phagocytosis.

CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection.

The Epstein‐Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV‐infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme‐based and the other Fas (CD95)/Fas ligand (FasL)‐based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8+ T‐cells increased in number, but CD56+ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell‐restricted intracellular antigens (TIA‐1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA‐1 positive cells was smaller than that in CAEBV. Fas‐positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL‐positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin‐ and/or granzyme‐positive cells was small in number in both CAEBV and IM cases. In double‐staining FasL and EBV in situ hybridization, FasL‐positive EBV‐infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV‐infected lymphocytes may evade immune attack through the expression of FasL.

Absence of cytotoxic molecules in CD8- and/or CD56-positive adult T-cell leukaemia/lymphoma.

Abstract Adult T-cell leukaemia/lymphoma (ATLL) cells usually exhibit a CD4+ (helper/inducer) phenotype (CD4+/8–/56–), and only a minority of tumours express the CD8 (cytotoxic/suppressor) or CD56 (natural killer [NK]-associated) antigens. TIA-1 is a cytotoxic granule-associated protein expressed in NK cells and cytotoxic T lymphocytes (CTLs). Granzyme B, perforin and Fas ligand (FasL) are also expressed in activated CTLs and NK cells. To clarify the cytotoxic potential of ATLL cells, immunohistochemistry was performed in CD8+ and/or CD56+ ATLL cells, using anti-TIA-1, anti-granzyme B, anti-perforin and anti-FasL antibodies. We studied nine cases of CD8+ and/or CD56+ ATLL, all of which exhibited monoclonal integration of human T-cell leukaemia virus type 1 (HTLV-1) proviral DNA. Four cases exhibited a CD8+/CD56– phenotype, four others had a CD8–/CD56+ phenotype, and one was CD8+/CD56+. All but one case also expressed the surface antigens CD3, TCR αβ, and CD4. Expression of granzyme B and TIA-1 were demonstrated in three and two cases, respectively, but none expressed perforin or FasL. In the control study, 10 cases with typical CD3+/4+/8–/56– ATLL demonstrated no expression of those cytotoxic-associated proteins. Our findings suggest that CD8 and/or CD56 positivity probably confer(s) no cytotoxic function on ATLL cells, and it is possible that CD8 and CD56 may be simply aberrant surface markers in ATLL.