Survival and clonal expansion of mutating "forbidden" (immunoglobulin receptor-deficient) epstein-barr virus-infected b cells in angioimmunoblastic t cell lymphoma.

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a peculiar T cell lymphoma, as expanding B cell clones are often present besides the malignant T cell clones. In addition, large numbers of Epstein-Barr virus (EBV)-infected B cells are frequently observed. To analyze the differentiation status and clonal composition of EBV-harboring B cells in AILD, single EBV-infected cells were micromanipulated from lymph nodes of six patients with frequent EBV(+) cells and their rearranged immunoglobulin (Ig) genes analyzed. Most EBV-infected B cells carried mutated Ig genes, indicating that in AILD, EBV preferentially resides in memory and/or germinal center B cells. EBV(+) B cell clones observed in all six cases ranged from small polyclonal to large monoclonal expansions and often showed ongoing somatic hypermutation while EBV(-) B cells showed little tendency for clonal expansion. Surprisingly, many members of expanding B cell clones had acquired destructive mutations in originally functional V gene rearrangements and showed an unfavorable high load of replacement mutations in the framework regions, indicating that they accumulated mutations over repeated rounds of mutation and division while not being selected through their antigen receptor. This sustained selection-free accumulation of somatic mutations is unique to AILD. Moreover, the survival and clonal expansion of "forbidden" (i.e., Ig-deficient) B cells has not been observed before in vivo and thus represents a novel type of viral latency in the B cell compartment. It is likely the interplay between the microenvironment in AILD lymph nodes and the viral transformation that leads to the survival and clonal expansion of Ig-less B cells.

A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation.

The biologic and pathologic features of B-cell malignancies bearing a translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 are still poorly described. Herein we report the results of a comprehensive cytogenetic, fluorescence in situ hybridization (FISH), molecular and histopathological survey of a large series of B-cell malignancies with t(14;19) or variant translocations. A total of 56 B-cell malignancies with a FISH-proven BCL3 involvement were identified with the translocation partners being IGH (n=51), IGL (n=2), IGK (n=2) and a non-IG locus (n=1). Hierarchical clustering of chromosomal changes associated with the t(14;19) indicated the presence of two different groups of IG/BCL3-positive lymphatic neoplasias. The first group included 26 B-cell malignancies of various histologic subtypes containing a relatively high number of chromosomal changes and mostly mutated IgVH genes. This cluster displayed three cytogenetic branches, one with rearrangements in 7q, another with deletions in 17p and a third one with rearrangements in 1q and deletions in 6q and 13q. The second group included 19 cases, mostly diagnosed as B-cell chronic lymphocytic leukemia (B-CLL), and characterized by few additional chromosomal changes (e.g. trisomy 12) and unmutated IgVH genes. In conclusion, our study indicates that BCL3 translocations are not restricted to B-CLL but present in a heterogeneous group of B-cell malignancies.

Antidisialoganglioside ricin A-chain immunotoxins show potent antitumor effects in vitro and in a disseminated human neuroblastoma severe combined immunodeficiency mouse model.

Several monoclonal antibodies (mAbs) were screened on different neuroblastoma cell lines to evaluate ricin A-chain immunotoxins for possible use against human neuroblastoma. Four mAbs were identified that exhibited high antitumor activity against neuroblastoma cell lines as measured in an indirect cytotoxicity assay. These mAbs, including 14G2a (antidisialoganglioside), ch14.18 (a humanized switch variant), BW704 (antidisialoganglioside), and chCE7 (anti-glycoprotein of M(r) 190,000), were subsequently linked via the bivalent linker N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-piridyldithio++ +)toluene to deglycosylated ricin A chain. The most potent immunotoxin, 14G2a.dgA, inhibited the protein synthesis of neuroblastoma cell lines IMR5 and NMB by 50% at concentrations of 6 x 10(-12) M. To test the antitumor efficacy of these immunotoxins in vivo, we developed a disseminated human neuroblastoma model in severe combined immunodeficiency mice. Treatment of tumor-bearing mice with 14G2a.dgA 12 days after tumor challenge resulted in a significant prolongation of survival as compared with phosphate-buffered saline-treated controls (16.8 versus 6.5 weeks). We conclude that ricin A-chain immunotoxins might be of potential use in the treatment of human neuroblastoma.

Altered expression of the retinoblastoma gene product in human high grade non-Hodgkin's lymphomas.

The retinoblastoma gene (RB) is a growth suppressor gene on the human chromosome 13q14. It encodes a 105 kDa phosphoprotein (p105), with DNA-binding capacity. P105 is thought to be involved in cell cycle control. Inactivation of RB is responsible for the development of retinoblastomas and occurs frequently in osteosarcomas and small cell lung cancer. In this study we looked at the RB-structure and expression in cell lines and primary lymphoma samples from patients with high grade non-Hodgkin's lymphoma (NHL). Forty five primary high grade NHL, the B-lymphoblastoid cell line IM-9 and the NHL cell line WSU-NHL were studied for RB structure by Southern blotting and for RB-expression by Northern blotting, Western blotting and immunocytochemistry. In all experiments freshly cryopreserved material was used. Southern and Northern experiments were performed with the 0.9 kb and 3.8 kb RB-cDNA probe. For the detection of p105 two different anti-p105-monoclonal antibodies were used in immunocytochemistry and Western blotting experiments. No RB mRNA and no p105 could be found in IM-9 cells. Twenty six high grade NHL samples (58%) showed no p105 expression. In the subgroup of centroblastic lymphomas 16 out of 21 and in Burkitt's lymphomas five out of eight showed no p105-expression. P105 expression is absent in 58% of high grade NHL, particularly in centroblastic and Burkitt's lymphomas, suggesting that inactivation of RB may play a crucial role in the pathogenesis of high grade NHL.

Selective recognition of rat follicular dendritic cells (dendritic reticulum cells) by a new monoclonal antibody Ki-M4R in vitro and in vivo.

Using unstimulated rat peritoneal cells as immunogen a new monoclonal antibody Ki-M4R was produced. Ki-M4R recognizes follicular dendritic cells (dendritic reticulum cells) in germinal centers of lymphoid follicles in lymphatic tissue. In addition, sinus lining cells, endothelia of postcapillary venules, as well as mesangial cells of the renal glomerula immunoreact with Ki-M4R in vitro as well as in vivo. This antibody might be useful for studying the interaction of follicular dendritic cells and B-cell immune response.

Ki-M2R, a new specific monoclonal antibody, discriminates tissue macrophages from reticulum cells and monocytes in vivo and in vitro.

Utilizing rat peritoneal macrophages as the immunogen, a new monoclonal antibody enabling differential monitoring of the mononuclear phagocyte system (MPS) by immunohistochemistry has been raised. Designated Ki-M2R, this antigen could be detected with the immune alkaline phosphatase reaction on all macrophages including those of bone marrow, lymphatic sinuses, lymphoid follicles, splenic red pulp, and von Kupffer cells of the liver, as well as on macrophages of connective tissue, renal interstitial tissue, serous cavities, and gastrointestinal tract. Langerhans cells--the MPS-derived reticulum cells of the epidermis--interdigitating reticulum cells, and dendritic reticulum cells of lymphoid follicles were invariably negative. Blood monocytes were rendered positive only after evolving into macrophages upon appropriate stimulation. Thus, Ki-M2R selectively labels monocytes after transformation into macrophages.

Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the 'Ki-67' proteins.

The monoclonal antibody (mab) Ki-67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new mabs Ki-S3 and Ki-S5 detect proliferating cells in frozen and paraffin-embedded tissues. They recognize proteins with the same molecular mass as Ki-67 in Western blot and for the first time also in immunoprecipitation experiments. With these mabs we were able to enrich and purify the Ki-67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA-deduced amino acid sequence already published for the Ki-67 proteins. Since we were able to immunoprecipitate the Ki-67 proteins, we performed various immunoprecipitation experiments to obtain more information about the nature of these proteins. After radiolabelling L428 cells with [35S]-methionine we were able to immunoprecipitate the Ki-67 proteins after only 5 min of labelling time. In turnover experiments the Ki-67 proteins could not be detected 3 h after the end of labelling. These data indicate a half-life of the Ki-67 proteins of about 90 min. Labelling experiments with [32P]-orthophosphate revealed that the Ki-67 proteins are phosphorylated. After dephosphorylation was blocked with okadaic acid or cell growth was arrested by means of Colcemid, the phosphorylation of the Ki-67 proteins was greatly increased, indicating that the Ki-67 proteins are phosphorylated via serine and threonine, and that the phosphorylation of the Ki-67 proteins increases in cycling cells. Labelling experiments with [3H]-mannose and [3H]-glucose revealed that the protein is weakly N-glycosylated.

Migration and activation pattern of specialized dendritic cells after heterotopic small bowel transplantation in a graft-versus-host model of the rat.

Besides specific cellular-mediated T cell responses, B cell related humoral responses have been demonstrated during the course of graft-versus-host disease after semiallogeneic transplantation of cellular antigen. Following semiallogeneic small bowel transplantation, there are, besides others, two specific forms of antigen-presenting cells, namely sinus lining cells (SLCs) and follicular dendritic cells (FDCs) which mediate primary and secondary humoral immune responses, respectively. This study was aimed to clarify the role of these dendritic cell entities after transplantation of small bowel grafts in a one-sided graft-versus-host (GvH) model for untreated and immunosuppressed (15-deoxyspergualin) recipient animals. As graft-versus-host disease progressed, SLCs and FDCs were eliminated in donor and recipient graft-versus-host associated target tissues (spleen and mesenteric lymph nodes) of untreated animals, whereas these dendritic cells prevailed in immunosuppressed recipients. 15-deoxyspergualin successfully prevented GvHD and significantly prolonged the mean survival time of untreated rats (16.0 +/- 4.5 d) for at least 21 d. Based on the immunosuppressive efficacy of 15-deoxyspergualin on the survival and function of SLCs and FDCs, an unaltered development of germinal centers and B cell proliferation within mesenteric lymph nodes and spleen was maintained

Immunophenotyping of dermal spindle cell tumors: diagnostic value of monocyte marker Ki-M1p and histogenetic considerations.

Various studies have reported the utility of anti-CD34 staining in the differential diagnosis of dermal spindle-cell tumors. To investigate whether monoclonal antibody Ki-M1p might add practical diagnostic information, we examined a total of 120 cutaneous spindle cell neoplasms using a panel of markers. Anti-CD34 antibody QBEnd/10 consistently stained dermatofibrosarcomas, Kaposi's sarcomas, neurofibromas, and, to a lesser extent, hemangiopericytomas. A positive reaction was also found in > 18% of the dermatofibromas. Ki-M1p staining showed an intense immunoreaction in all dermatofibromas, whereas no reactivity was observed in dermatofibrosarcomas. In addition, a subset of cells was labeled in atypical fibroxanthomas and Kaposi's sarcomas. Neurofibromas, spindle-cell hemangioendotheliomas, and hemangiopericytomas were negative. Dermatofibrosarcomas and atypical fibroxanthomas also moderately expressed smooth muscle-specific actin. Immunohistochemically, a discrimination between dermatofibrosarcomas and neurofibromas was possible only by means of an antibody against the nerve growth factor receptor. We conclude that the combination of several antibodies, in particular anti-CD34 and Ki-M1p, may improve the accuracy of diagnostic immunohistochemistry in the field of cutaneous spindle cell tumors. We speculate that dermatofibroma is primarily a macrophage-rich inflammatory lesion in which cytokine secretion induces a secondary proliferation of fibroblasts, whereas dermatofibrosarcoma is likely to issue from primitive dermal cells of uncertain origin.

Detection of human topoisomerase II alpha in cell lines and tissues: characterization of five novel monoclonal antibodies.

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the alpha-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase II beta was ruled out by testing the antibodies on crude extracts from yeast cells expressing the beta-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.

Immunohistochemical monitoring of plasmacytoid cells in lymph node sections of Kikuchi-Fujimoto disease by a new pan-macrophage antibody Ki-M1P.

The new monoclonal antibody Ki-M1P, which detects a formalin-resistant epitope on conventional paraffin sections, was applied in 20 cases of different stages of Kikuchi-Fujimoto disease. This new pan-macrophage immunoreagent detects plasmacytoid T cells, referred to as plasmacytoid cells, and renders a reliable delineation of these cells against other similar cell types, such as blasts of high-grade B- and T-cell lymphoma. Histiocytes as well as macrophages were strongly positive, and plasmacytoid cells showed a somewhat weaker and primarily granular, intracytoplasmic immunoreactivity. Plasmacytoid cells, being a diagnostic feature of the Kikuchi-Fujimoto disease, facilitate a clear distinction of this disease entity from large cell or high-grade lymphomas. These results may represent an additional argument favoring the histiocytic origin of plasmacytoid cells. Additionally, they may point to an immunohistochemical tool that facilitates the differential diagnosis between Kikuchi-Fujimoto disease, especially in early stages of the disease, and malignant lymphoma.

Histopathology and Immunocytochemistry of Lymph Node Biopsies in Chronic Lymphocytic Leukemia and Immunocytoma.

When lymph node biopsies of lymphocytic neoplasms are studied with subtle morphological and immunohistochemical methods meaningful information is obtained, that is not only of theoretical value, but which also relates to the clinical behavior ("leukemia," paraproteinemia, prognosis). Such an analysis allows us to differentiate three subtypes of chronic lymphocytic leukemia (diffuse, pseudofollicular and tumor forming) and to recognize two subtypes of immunocytoma (lympho plasmacytoid and lympho plasmacytic). The plasmacytoid-plasmacytic differentiation is usually not recognizable in blood smears. CLL cells show only IgM + IgD on their surfaces, whereas in immunocytoma monotypic cytoplasmic immunoglobulins are evident, and they are not only of the IgM type. The CD5 negative lymphoplasmacytic subtype differs clearly from chronic lymphocytic leukemia and from the majority of cases of lymphoplasmacytoid immunocytoma, which are CD5 positive.

Cutaneous monoblastic leukemia as a first sign of relapse six years after autologous bone marrow transplantation for acute leukemia.

A patient with acute monoblastic leukemia (AML, M5A) was treated successfully in December 1987. In 1993 after 6 years in complete remission, she presented with an intracutaneous nodular mass on her right upper arm which was resected in toto and shown to be undifferentiated monoblastic leukemia. Two further chloroma lesions were excised in July 1994 and March 1995 respectively. Bone marrow cytology and histology always showed a continuing complete remission with no evidence of leukemia relapse. In July 1995 she presented with a disseminated skin infiltrate and a relapse with 80% monoblasts in the bone marrow. After one course of chemotherapy (Idarubicin/Ara-C), a second complete remission was achieved and her leukemic skin infiltrate disappeared completely. This case illustrates that chloromas of the skin can occur as late as 6 years after treatment for AML and also emphasizes that the occurrence of a chloroma does not necessarily mean immediate leukemia relapse. It also stresses that a second complete remission can be achieved with standard AML-induction therapy despite widespread leukemic skin infiltrates in such patients.

Follicular dendritic cells in non-Hodgkin's lymphomas.

Follicular dendritic cells (FDC) are restricted to the B-cell regions of secondary lymphoid tissue and to non-Hodgkin's lymphomas derived from the follicular center or the mantle zone. With their cytoplasmic ramifications they form a dense network which contains the B-lymphocytes. In situ, FDC are only detectable at the ultrastructural level or when stained with anti FDC-reagents. On the surface of their dendritic extensions they express transferrin receptors (CD71), the B-cell epitope CD20, class II antigens, the myelomonocytic molecule CD14, the glycoprotein gp50 (CD40), and several receptors for components of the complement system (CD11b, CD21, CD35). Subsequent to an antigen challenge, FDC trap and retain immune-complexes for a long period of time. In vitro FDC and neoplastic lymphocytes spontaneously form small cellular aggregates. This adhesion is mediated by the LFA-1-alpha/beta = ICAM-1, the VLA-4 = VCAM-1, and the ICAM-1 = C3bi- receptor ligand pathways on B-cells and on FDC, respectively. The loss of LFA-1- alpha/beta and ICAM-1 molecules may enable neoplastic lymphocytes to detach from FDC. The monoclonal B-cells now invade new compartments. In vitro, FDC have the capacity to activate resting B-cells and to save them from dying by apoptosis. Signals involved in this activation include cell-surface immunoglobulin and CD40. Immunocytochemistry and autoradiography with single cell suspensions of neoplastic B cells suggest that FDC also provide signals leading to the continued stimulation of lymphoma lymphocytes. During the early stage of HIV infection lymph nodes show an immense follicular hyperplasia, with a massive increase of the dendritic network of FDC. In the later stage of the disease, the continuous involution of the germinal centers is associated with a progressive destruction of FDC.

[Castleman's disease: evaluation of 338 cases]. / Morbus Castleman: Auswertung von 338 Fällen.

PURPOSE: To evaluate a large group of patients with Castleman's tumours (Castleman's disease = MC) with regard to age, sex and tumour localisation. PATIENTS AND METHODS: 338 cases of MC were evaluated. All cases were registered by the Lymphknotenregister of the German Society of Pathology, Kiel. RESULTS: 292 MC of the hyaline vascular type (MC-HV) and 46 of the plasma cell type (MC-PC) were found, 259 (76.6%) in nodal and 79 (23.4%) in extranodal localisation. Main nodal localisation were the neck (25.5%) and the axilla (12.8%). The mediastinum was involved in only 6.2%. There was a slight preference of female (56%) compared to male (44%). Patients with MC-PC (mean age: 50 +/- 16.9 years) were significantly older (p < 0.001) than patients with MC-HV (mean age: 39 +/- 18.9 years). CONCLUSIONS: The main localisation of MC is the neck. MC-HV is more common than MC-PC. There is a slight preference for women. Mean age of patients with MC-PC is approximate 10 years higher than for MC-HV.

Size and composition of liver vitamin A reserves of human beings who died of various causes.

Postmortem livers from 77 "normal" persons, 37 patients with neoplastic disease, 10 subjects with liver insufficiency, and 7 infants were analysed for free and esterified retinol. The average concentrations of total vitamin A for the members of each group were 597, 551, 289, and 162 micrograms/g wet liver, respectively. Compared with corresponding control values, both cancer victims and patients with liver disease had significantly lower hepatic vitamin A levels. With regard to the composition of the liver vitamin A reserves, our results show that approximately 97% of this vitamin was present as retinyl ester. Additionally, minute amounts of retinol were also found in most of the liver specimens analysed. In "normal" subjects the major ester fraction recovered was palmitate/oleate followed by stearate and myristate/linoleate. By contrast, the second most abundant fatty acid in the retinyl ester fraction of cancer victims was myristic/linoleic acid together with significantly smaller quantities of stearic acid. In tissue samples obtained from patients with liver disorders, however, the myristate/linoleate fraction was increased and therefore nearly equal amounts of both retinyl stearate and myristate/linoleate were present.

[Follicular and mantle cell lymphoma. Extranodal involvement of primarily nodal indolent B-cell lymphomas]. / Follikuläre Lymphome und Mantelzelllymphome. Extranodale Manifestationen primär nodaler indolenter B-Zell-Lymphome.

Indolent (low-grade) B-cell non-Hodgkin's lymphomas, such as follicular and mantle cell lymphomas, are primarily nodal lymphomas in which lymph node involvement is the dominant clinical feature. The frequency of extranodal manifestations of these primarily nodal lymphomas is often underestimated. The typical growth pattern of nodal lymphomas can be absent or not evaluable because the biopsy specimens are often small in cases with extranodal involvement. Therefore, the immunophenotype of the lymphoma cells is of great importance for the diagnosis of indolent lymphomas with extranodal localizations.