Hodgkin-derived Cell Lines Research Articles

Recurrent mutations of the STAT6 DNA binding domain in primary mediastinal B-cell lymphoma

AbstractPrimary mediastinal B-cell lymphoma (PMBL) is a separate entity of aggressive B-cell lymphoma, characterized by a constitutive activation of janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, also observed in Hodgkin lymphoma. Although many cancers exhibit constitutive JAK-STAT pathway activation, mutations of STAT genes have not been reported in neoplasms. Here, we show that MedB-1 PMBL-derived and L1236 Hodgkin-derived cell lines and 20 of 55 (36%) PMBL cases harbor heterozygous missense mutations in STAT6 DNA binding domain, whereas no mutation was found in 25 diffuse large B-cell lymphoma samples. In 3 cases, somatic origin was indicated by the absence of the mutations in the nontumoral tissue. The pattern of STAT6 mutations was different from the classical features of somatic hypermutations. The mutant STAT6 proteins showed a decreased DNA binding ability in transfected HEK cells, but no decrease in expression of STAT6 canonical target genes was observed in PMBL cases with a mutated STAT6 gene. Although the oncogenic properties of STAT6 mutant proteins remain to be determined, their recurrent selection in PMBL strongly argues for their involvement in the pathogenesis of this aggressive B-cell lymphoma.

Oncogenic association of the Cbp/PAG adaptor protein with the Lyn tyrosine kinase in human B-NHL rafts

B-non-Hodgkin lymphomas (B-NHLs) use a raft-associated signalosome made of the constitutively active Lyn kinase, the tyrosine phosphorylated Cbp/PAG adaptor, and tyrosine phosphorylated STAT3 transcription factor. No such "signalosome" is found in rafts of ALK(+) T lymphoma and Hodgkin-derived cell lines, despite similar Cbp/PAG, Lyn, and STAT3 expression and similar amounts of raft sphingolipids. Stable association of the signalosome with B-NHL rafts requires (1) a Lyn kinase (auto)phosphorylated in its regulatory and active site tyrosines, (2) a Cbp/PAG adaptor phosphorylated at tyrosine 317 and bound to Lyn SH2 via phosphotyrosine 299 and neighboring residues, and (3) a tyrosine phosphorylated STAT3 linked via SH2 to the regulatory, C-terminal tyrosine of Lyn. No Csk appears to be part of this B-NHL signalosome. An oncogenic role for Lyn was shown after exposure of B-NHL lines to Lyn inhibitors that prevented Lyn and Cbp/PAG phosphorylation, dissociated the signalosome from rafts, and eventually induced death. Cell death followed decreases in Lyn or Cbp/PAG expression levels in one mantle cell lymphoma line, but not in a Hodgkin-derived one. The Lyn-Cbp/PAG signalosome appears to control proliferation and survival in most B-NHLs and constitutes a therapeutic target in B-NHL cells that exhibit oncogenic "addiction" to the Lyn kinase.

Long-term follow-up study on the role of serum CA-125 as a prognostic factor in 221 newly diagnosed patients with Hodgkin's lymphoma

The present study has explored the possible value of sCA-125 as a prognostic factor in Hodgkin's lymphoma (HL). From August 1992 to June 2005 sCA-125 was measured at presentation and at the end of the treatments in 221 newly diagnosed adult patients with HL. In this study 90/221 (41%) patients showed a value greater than the standard upper limit of 35 U/ml, and 79/90 (88%) with an abnormal sCA-125 were at an advanced stage of the disease. Patients with elevated sCA-125 showed a significant reduction in complete remission (CR) rate (76%vs. 98%; p < 0.0001). Failure of normalization of sCA-125 during the treatment revealed that CR had not been reached. Furthermore, no traces of the glycoprotein sCA-125 were found in a series of paraffin-embedded samples coming from 15 patients of this study. In addition, soluble CA-125 was not detected in supernatants coming from four different Hodgkin-derived cell lines. The long-term follow-up revealed that the group of patients with sCA-125 lower than 35 U/ml, at diagnosis, had an estimated 92% event free survival (EFS) rate and a 94% overall survival (OS) rate, while the group of patients with sCA-125 greater than 35 U/ml had only a 60% EFS rate (log-rank 33.43, p < 0.0001) and a 70% OS rate (log-rank 23.52, p < 0.0001). Extranodal disease, severe lymphocytopenia and age proved to be the only standard factors that could represent a poor chance to survive. At multivariate analysis, high sCA-125, E sites >1 and age were the only independent factors producing poor outcomes in terms of CR, EFS and OS. Therefore, we believe that sCA-125 is a simple, reliable and reproducible tool, which may improve existing prognostic systems.

Activating Transcription Factor 3 (ATF3) Is Overexpressed in Classical Hodgkin Lymphoma and Is Required for Survival of Hodgkin and Reed-Sternberg Cells in Response to Cellular Stress.

Hodgkin and Reed-Sternberg (HRS) cells display aberrant activity of several transcription factors, including nuclear factor kappa B (NF-kB) and members of the activator protein-1 (AP-1) family, that promote cytokine expression, cellular survival and proliferation. Using oligonucleotide microarrays, we set out to identify additional factors which are responsible for the malignant phenotype of HRS cells. Microarray analysis and subsequent Northern and Western blotting revealed that activating transcription factor 3 (ATF3), a member of the CREB/ATF family of transcription factors that is involved in the cellular response to stress signals, shows a strong constitutive expression in Hodgkin-derived cell lines. In contrast, ATF3 expression could not be detected in several Non-Hodgkin cell lines of distinct differentiation stages, ranging from pre-B cell lines to plasma B cell lines. To investigate the expression pattern of ATF3 in primary tumor cells, we performed immunohistochemistry on tissue microarrays. Immunohistochemistry confirmed a strong expression of ATF3 in primary HRS cells with a nuclear staining pattern. ATF3 expression was observed in almost all cases of classical Hodgkin lymphoma examined (69/71), whereas ATF3 expression was not detected in lymphocyte-predominant Hodgkin lymphoma. In addition, a significant number of anaplastic large cell lymphomas revealed a nuclear ATF3 staining pattern. In contrast, among all other B and T cell Non-Hodgkin lymphomas, ATF3 expression was observed only in rare cases (7/244), indicating that ATF3 expression is almost exclusively restricted to classical Hodgkin lymphoma and to a subset of anaplastic large cell lymphomas. To investigate the role of ATF3 for proliferation and survival of HRS cells, we generated vector-based siRNA constructs to downregulate ATF3 expression. Our vector system carries a puromycin-resistance gene thus allowing selection of siRNA expressing cells. Transfection of Hodgkin-derived cell lines with ATF3 siRNA constructs and subsequent selection of siRNA-expressing cells demonstrated a dramatic loss of viable cells. Our observations indicate that ATF3 is required for the survival of HRS cells exposed to the environmental stress imposed by antibiotic selection.

A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells

Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.

Constitutive Expression of c-FLIP in Hodgkin and Reed-Sternberg Cells

Crosslinking of the transmembrane receptor CD95/Fas leads to activation of a signaling cascade resulting in apoptosis. c-FLIP is a recently described protein that potently inhibits Fas-mediated apoptosis and has been shown to be a key factor in germinal center B cell survival. Because Hodgkin and Reed-Sternberg cells in classical Hodgkin's disease (cHD) are also resistant to Fas-mediated apoptosis we studied the role of c-FLIP in classical HD. High levels of c-FLIP protein were identified in two Fas-resistant Hodgkin-derived cell lines. In contrast to other tumor cells, inhibition of protein synthesis by cycloheximide did not lead to down-regulation of c-FLIP protein in these HD cell lines. Furthermore, Fas-mediated apoptosis was only partially restored suggesting that normal regulation of c-FLIP was disrupted. The in vivo relevance of these findings was supported by demonstration of significant c-FLIP expression by immunohistochemistry in 18 of 19 evaluable cases of primary HD. Taken together, c-FLIP is constitutively expressed in HD and may therefore be a major mechanism responsible for Fas-resistance in HD.

Tissue inhibitor of metalloproteinases 1 is an autocrine and paracrine survival factor, with additional immune-regulatory functions, expressed by Hodgkin/Reed-Sternberg cells

Tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 are proteins with proteinase-inhibiting and cytokine properties. TIMP-1 is active primarily in B cells and B-cell lymphomas, whereas TIMP-2 expression is restricted to T cells. The expression of TIMP-1 and TIMP-2 in lymph nodes from patients with Hodgkin disease (HD) and in Hodgkin-derived cell lines was investigated. In situ hybridization showed TIMP-1 RNA expression in 3% to 80% of Hodgkin/Reed-Sternberg (H/R-S) cells from 14 of 15 patients, with results in one patient being at the lowest detection limit; no expression of TIMP-2 in H/R-S cells; and only weak expression of TIMP-2 in reactive lymphoid tissue. Production of TIMP-1 protein by H/R-S cells was accordingly found on immunohistochemical analysis of lymph nodes from patients with HD. There was only low expression of matrix metalloproteinase (MMP)-2, which is mainly inhibited by TIMP-2; no expression of MMP-1 and MMP-3 in reactive lymphoid tissue; and no expression of these MMPs in H/R-S cells. Thus, TIMP-1 expression in lymph nodes was not correlated with metalloproteinase expression. Five of 7 Hodgkin-derived cell lines expressed TIMP-1 at the protein level. Only one of these cell lines expressed TIMP-2, at the lowest detection limit. TIMP-1 levels in plasma from patients with HD were within the same range as those in plasma from healthy controls. Recombinant human TIMP-1 inhibited induced cell death in Hodgkin-derived cell lines in vitro. TIMP-1 and TIMP-2 inhibited T-cell cytotoxicity against autologous cells presenting tumor-associated antigens and in allogeneic mixed lymphocyte cultures. Thus, TIMP-1, aside from its role in proteinase equilibrium, is an autocrine and paracrine survival factor for H/R-S cells and an immunosuppressive protein expressed in Hodgkin lymphomas.

Inhibition of metalloproteinases enhances the internalization of anti-CD30 antibody Ki-3 and the cytotoxic activity of Ki-3 immunotoxin.

CD30 is selectively expressed on the tumor cells of a variety of malignant disorders of the immune system and can therefore be used as a target for an anti-CD30 antibody-based immunotherapy. However, CD30 is cleaved at the cell surface by tumor necrosis factor-alpha converting enzyme (TACE). This metalloproteinase releases the soluble ectodomain of CD30 (sCD30), which is able to neutralize immunotherapeutic agents before these reach their target cells. Such constitutive CD30 cleavage is enhanced after binding of most anti-CD30 antibodies, leading to a downregulation of CD30 and an increase of sCD30 in the cell environment. Here, we demonstrate that CD30 shedding from the cell line Karpas 299 could effectively be blocked by the hydroxamic acid-based metalloproteinase inhibitors BB-3644 (IC50 = 180 nM), BB-2116 (IC50 = 230 nM), BB-94 (batimastat, IC50 = 230 nM) and BB-2516 (marimastat, IC50 = 1 microM). This inhibition reduced the concentration of sCD30 in the cell environment to the background level, prolonged the persistence of the anti-CD30 antibody Ki-3 on Karpas 299 cells and favored its internalization. Moreover, a nontoxic concentration of the inhibitor BB-3644 significantly increased the cytotoxic activity of the anti-CD30 ricin A-chain immunotoxin Ki-3.dgA towards the CD30(+) Hodgkin-derived cell line L540. Hence, the metalloproteinase inhibitor BB-3644 may be a promising compound to improve the immunotherapy of CD30(+) malignancies.

An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines.

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

Recombinant immunotoxins for the treatment of Hodgkin's disease (Review).

In recent years, substantial experience has been accumulated with tumor-specific immunotherapeutics which seem to be effective against minimal residual disease. The coupling of toxins to monoclonal antibodies has indicated promising results in early clinical trials. Recombinant DNA technology makes it possible to genetically fuse coding regions of V genes or cytokines to modified toxin domains. These recombinant immunotoxins can easily be manipulated to increase the cytotoxic potency or affinity. Binding single-chain variable fragments (scFv) expressed as chimeric fusion proteins on the surface of filamentous bacteriophages were selected on Hodgkin-derived cell lines. This technique was also used to create a new humanized anti-CD30 scFv which exhibits similar binding to the CD30 antigen when compared to its murine predecessor. ScFvs were then inserted into a new bacterial expression vector and thus fused to a deletion mutant of Pseudomonas exotoxin. Anti-CD25(scFv)-ETA' and anti-CD30(scFv)-ETA' were isolated from E. coli periplasm and purified by metal chelate affinity and size exclusion chromatography. All immunotoxins produced showed specific cytotoxicity against Hodgkin lymphoma cell lines as documented by competitive assays. In addition, these constructs were highly efficient in the treatment of disseminated human Hodgkin's disease in SCID mice. These in vivo data indicate a possible clinical impact for patients with relapsed CD25- and/or CD30-positive lymphoma.

An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line.

The human CD30 receptor is highly overexpressed on the surface of Hodgkin Reed-Sternberg cells and has been shown to be an excellent target for selective immunotherapy using monoclonal antibody-based agents such as immunotoxins. To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin's lymphoma, we have chosen the murine anti-CD30 hybridoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv). Hybridoma V-genes were polymerase chain reaction-amplified, assembled, cloned and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv were obtained by selection of binding phage on the Hodgkin lymphoma-derived, CD30-expressing cell line L540Cy. The selected recombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody. The Ki-4 scFv was subsequently fused to a deletion mutant of Pseudomonas exotoxin A (ETÁ). The resulting immunotoxin Ki-4(scFv)-ETÁ specifically binds to CD30+ L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM. This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgkin's lymphoma or other CD30+ malignancies. © 1999 Cancer Research Campaign

Construction and in vitro evaluation of RFT5(scFv)-ETA', a new recombinant single-chain immunotoxin with specific cytotoxicity toward CD25+ Hodgkin-derived cell lines.

The data of a closed phase I/II trial in patients with resistant Hodgkin's lymphoma indicate promising results using a chemically linked anti-CD25 ricin-A immunotoxin (IT) (RFT5-SMPT-dgA). This IT is based on the high-affinity moab RFT5. Since recombinant DNA technology permits the readier production of large amounts of ITs, we constructed a new RFT5-based fusion toxin [RFT5(scFv)-ETA']. We isolated mRNA from the hybridoma cell line RFT5, synthesized first strand cDNA and performed RT-PCR. Amplified coding regions of the light and heavy chain variable domains were joined together with a synthetic (Gly4-Ser)3 linker. The resulting single chain variable fragment (scFv) was fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-induced expression in Escherichia coli, the 70 kDa His-tagged fusion protein [RFT5(scFv)-ETA'] was isolated by osmotic shock and sonication under denaturing conditions. The recombinant toxin was purified on a Ni2+-NTA chelating sepharose and eluted with 250 mM imidazole. Pooled protein was renatured, dialyzed and concentrated by precipitation. Binding properties of RFT5(scFv)-ETA' were assessed on the CD25-expressing cell line L540cy by ELISA, immunohistochemistry and FACS analysis. CD25-specific binding was confirmed by immunoprecipitation experiments with recombinant human IL-2 receptor alpha. The in vitro toxicity of the chimeric protein was tested on the Hodgkin-derived cell lines L540cy, L428, L1236, a monocyte cell line U937 and a Burkitt lymphoma cell line BL38. RFT5(scFv)-ETA' inhibited protein biosynthesis of L540cy and L428 cells by 50% at concentrations (IC50) of 18 and 12 ng/ml, respectively. CD25-specific toxicity was confirmed by competitive toxicity assays. These data confirm for the first time binding specificity and toxicity of a recombinant anti-CD25 immunotoxin, against Hodgkin-derived cell lines; its applicability on Hodgkin's lymphoma needs yet to be evaluated in vivo.

Cure of disseminated xenografted human Hodgkin's tumors by bispecific monoclonal antibodies and human T cells: the role of human T-cell subsets in a preclinical model

Cure of a single established human Hodgkin's tumor growing subcutaneously in severe combined immunodeficient (SCID) mice can be achieved with a complex protocol using two bispecific monoclonal antibodies (Bi-MoAb) directed against the Hodgkin's associated CD30 antigen and the T-cell triggering molecules CD3 and CD28, respectively, together with human T cells prestimulated in vitro with Bi-MoAbs in the presence of CD30+ cells. To adapt this model to the clinical situation, disseminated tumors were established in SCID mice by intravenous injection of 2 x 10(7) cells of the Hodgkin's derived cell line L540CY. Treatment of SCID mice bearing disseminated CD30+ Hodgkin's tumors with the combination of CD3/CD30 and CD28/CD30 Bi-MoAbs and naive (ie, not in vitro prestimulated) human T cells resulted in the cure of all appropriately treated animals. T lymphocytes obtained from patients with advanced stage untreated Hodgkin's disease were as effective as lymphocytes from healthy controls. Treatment was effective even when delayed until 2 weeks after tumor inoculation, and application of Bi-MoAbs into SCID mice with circulating human T cells was as effective as injecting the Bi-MoAbs before the lymphocytes. Treatment results with isolated CD4+ and CD8+ human T cells suggest that both subsets are necessary for the Bi-MoAb mediated cure of xenografted human tumors in vivo. The efficacy and practicability of this preclinical immunotherapy protocol support and form the basis for the clinical evaluation of this approach in patients with Hodgkin's disease resistant to standard therapy.

Opposite effects of the CD30 ligand are not due to CD30 mutations: Results from cDNA cloning and sequence comparison of the CD30 antigen from different sources

CD30L, the ligand for the activation antigen CD30, is a member of the tumor necrosis factor family of cytokines. Binding of CD30L to CD30, which is a member of the nerve growth factor/tumor necrosis factor receptor family, induces proliferation in peripheral blood lymphocytes and Hodgkin's derived cell lines with a T-cell phenotype such as HDLM-2 and L540, while cell lines derived from anaplastic large cell lymphomas, such as Karpas 299, undergo cell death. In order to investigate whether mutations of the CD30 antigen are responsible for these opposite effects, we cloned the open reading frame of CD30 cDNAs from the cell lines L540 and Karpas 299 and from peripheral blood lymphocytes by reverse transcriptase polymerase chain reaction. Sequencing of independent plasmid clones revealed that these cells have a silent transition (A→G) at position 771 of the open reading frame compared to the published sequence derived from the HTLV-1 + cell line HUT-102. As published data have shown that crosslinking of CD30 induces an elevation of cytosolic free calcium ([Ca 2+] i) in TCR positive Jurkat cells, we have analysed the effect of crosslinking of CD30 on L540 and Karpas 299 cells. No elevations of [Ca 2+] i have been observed in these cell lines after crosslinking of CD30 with HRS-4. We conclude (i) that the different functional effects of CD30 in PBL, L540 and Karpas 299 are not due to differences in the primary structure of the receptor; and (ii) that the different responses observed upon engagement with CD30L for the cell lines L540 and Karpas 299 do not correlate with differences in mobilization of [Ca 2+] i after crosslinking of CD30.

Disseminated growth of Hodgkin's-derived cell lines L540 and L540cy in immune-deficient SCID mice

Local tumor growth has been reported after subcutaneous and intraperitoneal injection of Hodgkin's disease (HD) derived cell lines into different immunodeficient mouse strains. An animal model with disseminated growth of tumor cells would be useful for studying the in vivo biology of HD cells as well as for preclinical testing of new therapeutic regimens. For this purpose the HD-derived cell lines L540, L540cy, L428, and KM-H2 were injected intravenously into SCID mice. In contrast to L428 and KM-H2, widespread neoplasia occurred after a period of four to six weeks following injection of L540 and the subline L540cy. Lymph nodes were found to be the preferred site of tumor growth. CD30 surface antigen expression on Hodgkin cells and the karyotype of the tumor cells were preserved in the animal host. Thus, to a large extent, the SCID mouse model mimics the dissemination pattern of Hodgkin's disease in man. To evaluate the role of adhesion molecule expression in the dissemination of HD-derived cell lines, CD44 and members of the immunoglobulin, integrin, selectin, and Fc receptor families were quantified by flow cytometry. CD30 expression was also measured. Although CD44 expression has been correlated with dissemination in non-Hodgkin's lymphoma (NHL), this was not the case in the Hodgkin's SCID mouse model. CD44 was not expressed on the disseminating cell lines L540 and L540cy but was expressed in the nondisseminating lines L428 and KM-H2.

A CD16/CD30 bispecific monoclonal antibody induces lysis of hodgkin's cells by unstimulated natural killer cells In AND In vivo

In order to target NK cells against the Hodgkin's-derived cell line L540, we developed bispecific monoclonal antibodies (Bi-MAbs) by somatic hybridization of the 2 mouse hybridoma cell line HRS-3 and A9 which produce monoclonal antibodies (MAbs) with reactivity against the Hodgkin and Reed-Sternberg cell-associated CD30 antigen and the CD16 antigen (Fc gamma III receptor), respectively. The CD16 MAb-producing cell line A9 was selected as a partner for HRS-3 because of its efficiency in inducing lysis of the A9 hybridoma cells by resting NK cells. The hybrid hybridoma cell line HRS-3/A9 produced the supernatant with the strongest bispecific reactivity and was repeatedly subcloned and used for ascites production. Crude supernatant and purified HRS-3/A9 Bi-MAb triggered specific lysis of the CD30+ Hodgkin's-derived cell line L540, but not of the CD30- cell line HPB-ALL by unstimulated peripheral-blood lymphocytes and NK-cell-enriched populations. Moreover, treatment of SCID mice bearing heterotransplanted human Hodgkin's tumors with HRS-3/A9 and human peripheral blood lymphocytes induced specific complete tumor regression in 10/10 animals. We thus report successful tumor treatment in an in vivo model using NK-cell-associated Bi-MAbs and show that the Bi-MAb HRS-3/A9 is an efficient promoter of the anti-tumor effects of NK cells in vitro and in vivo.

Hodgkin derived cell lines L540 and L540cy grow disseminatedly in severe combined immunodeficient (SCID) mice

Hodgkin derived cell lines L540 and L540cy grow disseminatedly in severe combined immunodeficient (SCID) mice

Clinical significance of soluble CD30 antigen in the sera of patients with untreated Hodgkin's disease

The soluble form of the CD30 antigen (sCD30), an 88-kd glycoprotein that is released by Hodgkin's-derived cell lines in vitro, can be detected in patients with Hodgkin's lymphoma, adult (HTLV-1+) T-cell leukemia, rare cases of non-Hodgkin's lymphoma, and acute infectious mononucleosis (anti-EBV-IgM+). In a prospective study of 90 consecutive untreated patients with newly diagnosed Hodgkin's disease who were treated according to the protocols of the German Hodgkin Study group, 22% had detectable levels of sCD30 in their serum. sCD30 was only detected in patients with B symptoms (20 of 44 or 45%), and maximum sCD30 levels (88 U/mL) were found in stage IVB. Of 87 patients evaluable for response, sCD30+ patients had significantly lower rates of complete remission (9 of 20 or 45% v 60 of 67 or 90%; P less than .001) and higher rates of progressive disease (9 of 20 or 45% v 6 of 67 or 9%; P less than .001) than CD30+ patients. Similarly, freedom from treatment failure curves were significantly worse for CD30+ patients (P = .0003). sCD30 disappeared after successful treatment, but increased in patients with progressive disease. It was never detected in patients in complete remission or in healthy controls. We conclude that sCD30 is a valuable marker for disease activity and has prognostic significance in Hodgkin's disease.

Clinical Significance of Soluble CD30 Antigen in the Sera of Patients With Untreated Hodgkin's Disease

The soluble form of the CD30 antigen (sCD30), an 88-kd glycoprotein that is released by Hodgkin's-derived cell lines in vitro, can be detected in patients with Hodgkin's lymphoma, adult (HTLV-1+) T-cell leukemia, rare cases of non-Hodgkin's lymphoma, and acute infectious mononucleosis (anti-EBV-IgM+). In a prospective study of 90 consecutive untreated patients with newly diagnosed Hodgkin's disease who were treated according to the protocols of the German Hodgkin Study group, 22% had detectable levels of sCD30 in their serum. sCD30 was only detected in patients with B symptoms (20 of 44 or 45%), and maximum sCD30 levels (88 U/mL) were found in stage IVB. Of 87 patients evaluable for response, sCD30+ patients had significantly lower rates of complete remission (9 of 20 or 45% v 60 of 67 or 90%; P less than .001) and higher rates of progressive disease (9 of 20 or 45% v 6 of 67 or 9%; P less than .001) than CD30+ patients. Similarly, freedom from treatment failure curves were significantly worse for CD30+ patients (P = .0003). sCD30 disappeared after successful treatment, but increased in patients with progressive disease. It was never detected in patients in complete remission or in healthy controls. We conclude that sCD30 is a valuable marker for disease activity and has prognostic significance in Hodgkin's disease.

Heterogeneous expression of proto‐oncogenes in Hodgkin's disease derived cell lines

The expression of 20 proto-oncogenes was analysed by Northern blotting in four cell lines derived from patients with Hodgkin's disease (L428, L540, CO and DEV) and compared to lymphoid and myeloid leukemia cell lines and normal hematopoietic cells. Expression of the proto-oncogenes c-myc, p53, c-jun, pim-1, lck, c-syn, c-raf and N-ras were detected in Hodgkin's disease derived cell lines and in normal hematopoietic cells. Transcripts of the proto-oncogene c-met were detected in the Hodgkin's derived cell lines L428 and L540 but not in the lymphoid or myeloid leukemia cell lines or in tonsil cells, peripheral blood mononuclear cells and granulocytes. Expression of the proto-oncogenes N-myc and lck were observed in the Hodgkin's derived cell line CO which express T cell receptor genes and in the T cell lines JM and CEM. L428 cells and CO cells expressed aberrant transcripts of the c-fes proto-oncogene. Thus Hodgkin's disease derived cell lines are heterogeneous in their expression pattern of proto-oncogenes, expressing normal and aberrant transcripts of proto-oncogenes which are not found in untransformed hematopoietic cells.