Bispecific antibody-mediated destruction of Hodgkin's lymphoma cells

CD30 is a molecule that is overexpressed on the surface of Hodgkin's lymphoma cells. Therefore, CD30 represents a potential candidate for immunotherapy. In this study, we report the in vitro results of two bispecific molecules (BSMs) that target CD30 to trigger molecules expressed on myeloid effector cells. The first BSM is composed of the Fab' fragment of a CD30-specific antibody, Ki-4, chemically linked to the Fab' fragment of the humanized CD64 (FcgammaRI)-specific antibody, H22 (H22xKi-4). In the second BSM, the H22 Fab' is replaced with the Fab' fragment of the CD89 (FcalphaR)-specific, antibody, A77 (A77xKi-4). Both BSMs were able to bind specifically to lymphoma cell lines expressing CD30. In addition, the H22xKi-4 and A77xKi-4 BSMs were shown to bind cells expressing CD64 and CD89, respectively. Both BSMs mediated potent, dose-dependent antibody dependent cell-mediated cytotoxicity (ADCC) of CD30-expressing tumor cell lines when human monocytes were used as effector cells. In addition, freshly prepared polymorphonuclear leukocytes (PMNs) and effector cells in whole blood were able to mediate the ADCC of targets in conjunction with the A77xKi-4 BSM in some, but not all, experiments. Furthermore, we examined the ability of monocyte-derived macrophages (MDMs) to phagocytose CD30-expressing tumor cell lines in conjunction with the BSM. MDM-mediated phagocytosis was significantly enhanced in the presence of both BSMs. These results demonstrate that targeting lymphoma cells via CD30 to the myeloid high affinity Fc receptor for IgG and to the Fc receptor for IgA results in potent in vitro anti-tumor activity.     

Expression of p63 in anaplastic large cell lymphoma but not in classical Hodgkin's lymphoma

Immunohistochemical determination of p63 protein is frequently used in the pathologic diagnosis of nonhematological solid tumors. In malignant hematological disease, p63 expression has been reported in 22% of follicular lymphoma, about 35% of diffuse large B-cell lymphoma, 23% of chronic lymphocytic leukemia, and in some cases of blast crisis of chronic myelogenous leukemia. Anaplastic large cell lymphoma is a rare disease that accounts for less than 5% of all cases of non-Hodgkin's lymphoma. There is little information concerning p63 expression in this specific type of lymphoma. In some cases, the morphological and phenotypic features between anaplastic large cell lymphoma and classical Hodgkin's lymphoma are similar, making this differential diagnosis challenging. We studied p63 expression using a tissue microarray approach in 154 cases of anaplastic large cell lymphoma, including 38% anaplastic large cell kinase positive and 62% anaplastic large cell kinase negative, and 58 Hodgkin's lymphoma cases. Sixty-eight cases of anaplastic large cell lymphoma (44%) showed p63 nuclear positivity (41% of anaplastic large cell kinase positive and 47% of anaplastic large cell kinase negative). Of 130 cases of systemic-anaplastic large cell lymphoma, 42% showed p63 positivity. The neoplastic cells expressed p63 in 38% of the cases of CD45-negative/anaplastic large cell kinase-negative null cell-type anaplastic large cell lymphoma, a subgroup that offers the most difficulties in the differential diagnosis with classical Hodgkin's lymphoma. In contrast, none of the cases of classical Hodgkin's lymphoma demonstrated any p63 expression. These results demonstrate that p63 protein expression is frequently expressed in a subset of anaplastic large cell lymphoma cases and may be used as a potential tool in the differential diagnosis between anaplastic large cell lymphoma and classical Hodgkin's lymphoma.     

Leu M1 and peanut agglutinin stain the neoplastic cells of Hodgkin's disease

It has been suggested that the malignant cells of Hodgkin's disease (HD), Reed-Sternberg cells, and their mononuclear variants may be related to cells of the monocyte-histiocyte system. To test this hypothesis, 20 cases of HD were tested with nine antibodies, monoclonal or polyclonal, that normally react with cells of monocytic/histiocytic/granulocytic lineages, as well as PNA, which binds to histiocytes directly. Only two reagents, Leu M1 and PNA bound to the neoplastic cells in 20/22 and 13/22 cases tested, respectively. Leu M1 was the most sensitive reagent and was negative in only two cases of lymphocyte predominant HD. Leu M1 also could be employed in routinely fixed and processed formalin or B5-fixed tissue. This antibody, which was negative in 27 cases of non-Hodgkin's lymphoma, including 12 peripheral T-cell lymphomas, should prove to be of value in differential diagnosis of HD and morphologically similar reactive and neoplastic lymphoid lesions.     

Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkin's lymphoma cells mediates Up-regulation of CCL20 and the migration of regulatory T cells

In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.     

Hodgkin's lymphoma: the pathologist's viewpoint

Despite its well known histological and clinical features, Hodgkin's lymphoma (HL) has recently been the object of intense research activity, leading to a better understanding of its phenotype, molecular characteristics, histogenesis, and possible mechanisms of lymphomagenesis. There is complete consensus on the B cell derivation of the tumour in most cases, and on the relevance of Epstein-Barr virus infection and defective cytokinesis in at least a proportion of patients. The REAL/WHO classification recognises a basic distinction between lymphocyte predominance HL (LP-HL) and classic HL (CHL), reflecting the differences in clinical presentation and behaviour, morphology, phenotype, and molecular features. CHL has been classified into four subtypes: lymphocyte rich, nodular sclerosing, with mixed cellularity, and lymphocyte depleted. The borders between CHL and anaplastic large cell lymphoma have become sharper, whereas those between LP-HL and T cell rich B cell lymphoma remain ill defined. Treatments adjusted to the pathobiological characteristics of the tumour in at risk patients have been proposed and are on the way to being applied.     

Composite lymphoma: the interface between Hodgkin's and non-Hodgkin's lymphoma

Hodgkin's lymphoma is a rare neoplasm mainly affecting young people. Within the last decade, nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) has been separated from all other cases of the so-called classical type. Moreover, the derivation of Hodgkin's lymphoma from germinal centre B-cells has been established, giving rise to a possible clonal relationship between Hodgkin's lymphoma and the non-Hodgkin's lymphomas (NHLs). The interface between them therefore has both diagnostic and biological aspects. Diagnostic difficulties arise, due to overlapping definitions, between biologically unrelated entities, e.g. classical Hodgkin's lymphoma and NLPHL or anaplastic large cell lymphoma, respectively. On the other hand, there is a biological grey zone regarding composite lymphomas consisting of Hodgkin's lymphoma and any NHL. This simultaneous or subsequent occurrence of Hodgkin's lymphoma and non-Hodgkin's lymphomas may, or may not, be clonally related. For management purposes lymphomas, e.g. NLPHL and T cell-rich B-cell lymphoma, must be distinguished along a continuous spectrum of progression.     

Expression of the chemokine receptor CXCR2 in normal and neoplastic neuroendocrine cells

BACKGROUND: Chemokines effect their proinflammatory and growth regulatory roles through interaction with serpentine receptors. One such receptor, CXCR2, binds multiple CXC chemokines, including interleukin 8, GRO-alpha, GRO-beta, GRO-gamma, and NAP-2. We have previously identified CXCR2 expression on myeloid cells, notably mature granulocytes, and projection neurons. OBJECTIVE: To determine the expression of CXCR2 by cells of the neuroendocrine system. DESIGN: Archival specimens from normal neuroendocrine tissues and their malignant counterparts were analyzed by immunohistochemistry with monoclonal antibodies specific for CXCR1 and CXCR2. RESULTS: Immunohistochemical analysis revealed high-level expression of CXCR2 by cells in the pituitary, adrenal medulla, pancreatic islets, thyroid C cells, scattered Kulchitsky cells in the bronchi, and counterpart neuroendocrine cells in the stomach, small bowel, colon, and appendix. Neuroendocrine neoplasms that demonstrated high-level CXCR2 expression included (1) primary carcinoids localized to the stomach, small bowel, colon, appendix, fallopian tube, ovary, and lung; (2) atypical carcinoids of the lung; (3) metastatic carcinoids; (4) pituitary adenomas; (5) pheochromocytomas; and (6) medullary carcinomas of the thyroid. Small cell lung carcinomas, large cell neuroendocrine carcinomas of the lung, small cell carcinoma of the cervix, Merkel cell carcinomas, neuroblastomas, and malignant melanomas lacked evidence of CXCR2 expression. CONCLUSIONS: The expression of CXCR2 by normal neuroendocrine cells and neoplastic counterparts that have retained phenotypic features of this differentiation program suggests that chemokines may play an important role in functions that are characteristic of this cell type. In addition, this raises the possibility that chemokines may modulate secretion of biologically active products of these cells and their neoplastic counterparts.     

Vascular endothelial growth factor (VEGF) is expressed by neoplastic Hodgkin-Reed-Sternberg cells in Hodgkin's disease

Vascular endothelial growth factor (VEGF) is involved in tumour angiogenesis, an important process for the growth and metastatic potential of solid tumours. Numerous studies have demonstrated up-regulation of VEGF at both mRNA and protein level in various tumours and a correlation with advanced stage and prognosis has been demonstrated in some cases. Limited information exists about its role in lymphoid malignancies and in particular, Hodgkin's disease. The present study examined the immunohistochemical expression of VEGF using the monoclonal antibody VG1 in a series of 61 cases of Hodgkin's disease, including both classical Hodgkin's disease and the nodular lymphocyte predominance variant, and correlated these results with microvessel density, using an anti-CD31 monoclonal antibody. In 41 cases (70.6%) of classical Hodgkin's disease and one of the three cases of nodular lymphocyte predominance Hodgkin's disease, the neoplastic Reed-Sternberg and Hodgkin cells expressed VEGF. The staining observed was cytoplasmic, either diffuse or with a focal paranuclear distribution. Macrophages were always positive, while reactive lymphocytes showed occasional positivity. A variable amount of strong extracellular staining was also observed in the tissue stroma and intravascular plasma staining was prominent. There was no statistically significant relationship between VEGF expression and the subtype of Hodgkin's disease or microvessel density. In vitro studies using the Reed-Sternberg cell lines L428 and KM-H2 were also performed in both normoxia and hypoxia and VEGF protein production was assessed by flow cytometry (FACS), immunoassay of cell culture supernatant, and RT-PCR. Analysis by FACS demonstrated a subset of cells in both cell lines reacting with VG1 and analysis of secreted VEGF (pg/ml per 1x10(6) cells) in cell culture supernatant confirmed the normoxic production in both cell lines and significant hypoxic induction (p<0.005). Additionally, both cell lines expressed VEGF mRNA, as demonstrated using the RT-PCR method. In conclusion, neoplastic cells express VEGF in Hodgkin's disease, as is the case in solid tumours, and this expression may be induced by hypoxia. The presence of VEGF in reactive macrophages and in the extracellular matrix might facilitate tumour progression.     

Cytogenetic evidence for the origin of neoplastic cells in CD5-positive marginal zone B-cell lymphoma

We describe an 87-year-old female patient presenting with a breast lump and axillary lymphadenopathy. Histological examination revealed the lump to be a CD5-positive extranodal marginal zone B-cell lymphoma (MZBCL) of mucosa-associated lymphoid tissue. Subsequent staging revealed disseminated disease including the head and neck region. Only 2 cases of CD5-positive MZBCL have undergone any form of cytogenetic analysis, and we report the first standard karyotype of such a case. This revealed partial trisomy 3,7q deletion and an additional marker chromosome. Notably, cells lacked the t(11;14) found at high frequency in mantle cell lymphoma and trisomy 12 found in B small lymphocytic lymphoma (B-SLL). These results, combined with the clinical, histological, and immunophenotypic picture, suggest a marginal zone origin for the neoplastic lymphocytes, rather than a relationship with mantle cell or small lymphocytic lymphoma.     

Expression of myelin protein genes in Schwann cells

Summary The expression of myelin protein genes in Schwann cells has been studied byin situ hybridization.³⁵S-UTP-labelled, antisense and sense RNA probes to the major protein Po, myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and proteolipid protein (PLP) were employed with paraffin-embedded sections, teased fibres and dissociated Schwann cells from sciatic nerves of rats. Teased fibres were also prepared from cervical sympathetic trunks. Po mRNA was strongly expressed in the mid-internodal perinuclear area of Schwann cell cytoplasm. The degree of signal appeared to be related to fibre size. MBP mRNA showed a diffuse pattern along the Schwann cell internode with a marked increase in grains at the paranodal cytoplasm, particularly in larger fibres. This distribution suggests that the paranodal area is a major site of insertion of MBP into myelin membrane. The expression of MAG and PLP mRNA was markedly lower than Po and MBP. Both mRNAs were localized in the perinuclear cytoplasm and showed a dependence on fibre size. No significant signal was present in Schwann cells associated with unmyelinated axons.In addition to providing data on the cellular expression of myelin protein genes, these studies have shown that teased fibres are invaluable in allowing the localization of low abundance mRNAs.     

单细胞显微切割术在霍奇金淋巴瘤中的应用

目的：应用单细胞显微切割技术分离和研究霍奇金淋巴瘤（Ｈｏｄｇｋｉｎ‘ｓ ｌｙｍｐｈｏｍａ,ＨＬ）瘤细胞,并检测其是否存在人巨细胞病毒（ｈｕｍａｎ ｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ,ＨＣＭＶ）感染。方法：采用单细胞显微切割技术从ＨＬ组织切片中获得其瘤细胞Ｒｅｅｄ－Ｓｔｅｒｎｂｅｒｇ／Ｖａｒｉａｎｔ （ＲＳ／Ｖ）细胞,提取ＤＮＡ后,利用ＰＣＲ扩增ＨＣＭＶ立即早期基因片段。结果：单细胞显微切割可将ＲＳ／Ｖ     

Expression of interleukin-1 in Reed-Sternberg cells and neoplastic cells from true histiocytic malignancies.

Interleukin-1, a peptide produced by monocytes, histiocytes, and interdigitating reticulum cells, plays an important role in the regulation of immune function. In this styde, we examined the production of interleukin-1 in 115 patients with a variety of human lymphomas by using a rabbit anti-interleukin-1 antibody and the immunoperoxidase technique. Interleukin-1 was detected in Reed-Sternberg cells from 20 patients with Hodgkin's disease as well as in neoplastic cells from 9 patients with true histiocytic lymphoma or malignant histiocytosis. In the other 86 cases, which included T- and B-cell lymphomas, no interleukin-1 could be detected. This result indicates a close relationship between Hodgkin's disease and true histiocytic malignancies and provides additional evidence to support our hypothesis that Reed-Sternberg cells are related to interdigitating reticulum cells.     

Immunophenotypic differentiation between neoplastic plasma cells in mature B-cell lymphoma vs plasma cell myeloma

Some non-Hodgkin lymphomas show marked plasmacytic differentiation. In such cases, it may be difficult to differentiate these lymphoma from plasmacytoma or myeloma, especially with limited diagnostic material. However, there may be immunophenotypic differences in the plasma cells in these disorders that distinguish them. This study characterizes the immunophenotypes of neoplastic plasma cells in 41 cases of B-lineage non-Hodgkin lymphoma and compares them with those in plasma cell myeloma. We found that plasma cells in lymphoma were significantly more likely to express CD19, CD45, and surface immunoglobulin and less likely to express CD56 than those in myeloma. We further show that CD 19 and CD56 expression can be used reliably to distinguish these entities. Myeloma-associated osseous lesions and solitary plasmacytoma of bone showed myeloma-like immunophenotypes. However, some extramedullary plasmacytomas showed lymphoma-like phenotypes, suggesting that, in reality, they may represent non-Hodgkin lymphomas with extensive plasmacytic differentiation.