Hodgkin's Disease Cell Research Articles

Signaling via the anti-CD30 mAb SGN-30 sensitizes Hodgkin's disease cells to conventional chemotherapeutics

SGN-30, a monoclonal antibody with activity against CD30+ malignancies, is currently in phase II clinical evaluation for treatment of Hodgkin's disease (HD) and anaplastic large cell lymphoma. The mechanisms underlying SGN-30's antitumor activity were investigated using cDNA array of L540 cells. SGN-30 treatment activated NF-kappaB and modulation of several messages including the growth regulator p21WAF1/CIP1 (p21) and cellular adhesion marker ICAM-1. p21 protein levels increased coincident with growth arrest and Annexin V/PI staining in treated HD cells. To determine if SGN-30-induced growth arrest would sensitize tumor cells to chemotherapeutics used against HD, L540cy and L428 cells were exposed to SGN-30 in combination with a panel of cytotoxic agents and resultant interactions quantified by the Combination Effects Method. Interactions between SGN-30 and all cytotoxic agents examined were additive or better. These in vitro data translated to increased efficacy of SGN-30 and bleomycin against L540cy tumor xenografts. In addition to direct cell killing, SGN-30 affects growth arrest and drug sensitization through growth regulating and proapoptotic machinery. Importantly, these data suggest that SGN-30 can enhance the efficacy of standard chemotherapies used to treat patients with CD30+ malignancies.

The NFATc1 transcription factor is widely expressed in white cells and translocates from the cytoplasm to the nucleus in a subset of human lymphomas

Stimulation of lymphoid cells via their surface receptors triggers signalling pathways that terminate in the nucleus, where they induce alterations in gene transcription. Nuclear factor of activated T cells (NFAT) transcription factors, involved in a major Ca2+-dependent signalling pathway, normally reside in the cytoplasm but re-locate to the nucleus when activation of the pathway (e.g. following ligation of antigen receptors) leads to their dephosphorylation. This study found that one member of the NFAT family (NFATc1/NFAT2) can be detected in routine biopsy samples, where it is seen in essentially all lymphoid cells, but is absent from the great majority of non-haematopoietic cells. An immunohistological evaluation of NFATc1 in almost 300 lymphomas showed that most neoplastic lymphoid cells also express NFATc1 as a cytoplasmic constituent, although it is absent in classical Hodgkin's disease and plasma cell proliferations. Of particular interest was the finding that NFATc1 was relocated to the nucleus in a minority of lymphoid neoplasms (usually diffuse large B-cell lymphomas or Burkitt lymphoma), presumably reflecting activation of the NFAT pathway. It would be of interest to correlate this feature with patterns of gene expression and also with prognosis, since it may identify a subset of human lymphoma that is distinct in its molecular and clinical features.

Elevation of plasma membrane permeability by laser irradiation of selectively bound nanoparticles

Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkin's disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.

The role of interleukin-3 in classical Hodgkin's disease

Classical Hodgkin's disease (HD) is a peculiar form of lymphoma characterized by a low frequency of tumor cells, the so-called Hodgkin (H) and Reed/Sternberg (RS) cells, embedded in a background of non-neoplastic (reactive) cells believed to be recruited and activated by H - RS cell-derived cytokines/chemokines. How these tumor cells can survive in such a seemingly hostile environment has confused researchers. We have previously identified interleukin (IL)-3 receptor (R) expression as a common feature of classical HD and unveiled the potential role of IL-3 as a growth and anti-apoptotic factor for H - RS cells. More then 90% of malignant cells of classical HD usually express the α chain of the IL-3R (IL-3Rα), as evidenced by immunostaining of frozen sections and cell suspensions from neoplastic lymph nodes. Consistently, HD-derived cell lines (L428, KMH2, HDLM2 and L1236) express the α and β chains that form IL-3R, both at the mRNA and protein level, with a molecular size of IL-3Rα identical (70 kDa) to that expressed by human myeloid cells. Exogenous IL-3 promotes the growth of cultured H - RS cells, such an effect being potentiated by IL-9 and stem cell factor (SCF) co-stimulation, and is able to partially rescue tumor cells from apoptosis induced by serum deprivation. Finally, cultured H - RS cells are able to increase the production of IL-3 by pre-activated T cells, suggesting an involvement of IL-3/IL-3R interactions in the cellular growth of HD through paracrine mechanisms. This review will outline the biological activity of IL-3 and summarize the evidence indicating IL-3 as a growth and anti-apoptotic factor for H - RS cells in classical HD.

The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells

AbstractThe CD150 receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. A TxYxxV/I motif in the CD150 cytoplasmic tail can bind different SH2-containing molecules, including tyrosine and inositol phosphatases, Src family kinases, and adaptor molecules. To analyze CD150-initiated signal transduction pathways, we used DT40 B-cell sublines deficient in these molecules. CD150 ligation on DT40 transfectants induced the extracellular signal-regulated kinase (ERK) pathway, which required SH2-containing inositol phosphatase (SHIP) but not SH2 domain protein 1A (SH2D1A). CD150-mediated Akt phosphorylation required Syk and SH2D1A, was negatively regulated by Lyn and Btk, but was SHIP independent. Lyn directly phosphorylated Y327 in CD150, but the Akt pathway did not depend on CD150 tyrosine phosphorylation and CD150-SHP-2 association. Analysis of CD150 and SH2D1A expression in non-Hodgkin and Hodgkin lymphomas revealed stages of B-cell differentiation where these molecules are expressed alone or coexpressed. Signaling studies in Hodgkin disease cell lines showed that CD150 is linked to the ERK and Akt pathways in neoplastic B cells. Our data support the hypothesis that CD150 and SH2D1A are coexpressed during a narrow window of B-cell maturation and SH2D1A may be involved in regulation of B-cell differentiation via switching of CD150-mediated signaling pathways. (Blood. 2004;104:4063-4070)

Expression of Homeobox Gene HLXB9 Mediated by Constitutive Active Signal Transduction Pathways Activates IL-6 in Hodgkin's Disease Cell Lines.

Hodgkin's disease (HD) is characterized clinically by rare neoplastic giant cells surrounded by a mass of infiltrating diverse lymphocytes indicating the importance of cell communication for this disease. A variety of cytokines have been detected in HD patients and cell lines, including IL-6 which plays an important role in pathogenesis, and high levels of which are associated with bad prognosis. Regulators of IL-6 expression known to be actively overexpressed in HD cells are NFkB and AP-1 proteins. Recently, the homeobox protein GBX2 has been described to activate IL-6 in prostate carcinoma. Hence, to investigate IL-6 regulation in HD cells, we screened a panel of HD cell lines (HDLM-2, KM-H2, L-1236, L-428, L-540, SUP-HD1) by RT-PCR for expression of EHG family homeobox genes comprising GBX2, GBX1, EN1, EN2 and HLXB9. We found HLXB9 to be expressed in 5/6 HD cell lines correlating with IL-6 expression while GBX2 expression was absent. Inhibitory treatment of HD cell lines HDLM-2 and KM-H2 with antisense oligonucleotides (AS) directed against HLXB9 reduced both HLXB9 and IL-6 expression as detected by RT-PCR. HD cell line L-540 expressed neither HLXB9 nor IL-6 and, in contrast to other HD cell lines, lacked constitutive NFkB activity. NFkB activation by TPA treatment induced IL-6 expression in L-540, while combination of TPA treatment with forced expression of full length HLXB9 significantly enhanced IL-6 expression in these cells. HELA cells (which expressed HLXB9 but not GBX2) transfected with an IL-6 promoter-driven reporter construct (pIL6-EGFP) yielded green cells (30%). Treatment of similarly transfected HELA cells with AS-HLXB9 or transfection of HELA with pIL6-EGFP lacking the putative HLXB9 binding site showed significantly fewer green cells (20% and 22%, respectively). Taken together, these results suggest that HLXB9 directly enhances IL-6 expression in HD cells and may contribute to their unique phenotype. Recently, we identified ectopic expression of HLXB9 following chromosomal juxtaposition with MYB in acute myeloid leukemia (manuscript submitted). In contrast, we failed to detect chromosomal rearrangements of HLXB9 or MYB in HD cell lines. Expression of GBX2 in chicken AML cells requires both MYB and an activated signal transduction pathway. To investigate such a mechanism in HD cell lines which we had confirmed to express MYB protein, we pharmacologically inhibited certain signal transduction pathways (MAPK, JAK-STAT and PI3K) showing that all three pathways contribute to HLXB9 expression as analyzed by RT/RQ-PCR. Because MAPK and JAK-STAT pathways were recently shown to be constitutively activated in HD we performed AKT/phospho-AKT analysis to demonstrate constitutively active PI3K pathway in 5/6 HD cell lines. Thus, constitutive activated signal transduction pathways (probably together with MYB) contribute to HLXB9 expression in HD cell lines. In summary, we identified ectopic expression of the homeobox gene HLXB9 activating IL-6 expression in HD cell lines. The PI3K pathway was found to be permanently active in HD cell lines contributing together with other constitutive active signal transduction pathways to HLXB9 expression.

Anti-CD45 Mediated Cytolysis of Human T-Cell Lymphoma Cells.

Anti-CD45 mediated cytoreduction is an effective means for T-cell depletion in rodents and humans. In man, the CD45-specific rat monoclonal antibodies YTH24 and YTH54 are IgG2b subclass, exert a predominantly complement-dependent cytolytic activity against normal T-lymphocytes, and have been safely given to patients as part of conditioning therapies for allogeneic stem cell transplantation. The efficacy of such antibodies against human lymphoma is unknown. Therefore, we evaluated the cytolytic activity of YTH24 and YTH54 by complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), as well as by direct apoptotic and antiproliferative effects, against a panel of Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) cell lines, and against primary specimens. Significant CDC activity (>50% cytolysis) of the antibodies YTH54 and YTH24 was observed against three of five T-cell lymphoma lines, but against only one of nine B-cell lymphoma lines and none of four HD cell lines. The combination of YTH54 and YTH24 induced ADCC in all T-cell lymphoma cell lines and three primary leukemic T-cell lymphoma specimens, but were ineffective in B-cell lymphoma and HD cell lines.There were only minor effects of either antibody or the combination on lymphoma cell apoptosis or cell cycle arrest. In summary, anti-CD45 mediated CDC and ADCC via the antibodies YTH24 and YTH54 are primarily effective against lymphoma cells with T-cell phenotype, and may be an immunotherapeutic tool for the treatment of human T-cell lymphoma.

Latent-Membrane 2a Specific CTL's but Not Latent-Mebrane 1 Specific CTL's Lysis Efficiently Hodgkin Lymphoma Cells.

Relapse is the leading cause of treatment failure after allogeneic SCT of Hodgkin Disease (HD). As Ebstein-Barr infection (EBV) is associated with 60% of all HD cases, adoptive immunotherapy with donor derived EBV-specific T-cells lines has resulted in disease control of allogeneic SCT. Potential targets for the adoptively transferred T-cells are the type II latency protein LMP-1 and LMP-2a, which are both homogenously expressed by HD cells. In healthy individuals, both LMP-1 and LMP-2a elicits subdominant CD8+ T-cell responses with frequencies of less than 1:10000. LMP-1 and LMP-2a specific T-cells from 1x108 PBMC derived from HLA A*0201+healthy donors were stimulated with the HLA A*0201 LMP1-epitopes YLLEMLWRL and YLGQNWWTL and the HLA A*0201 LMP-2a epitope CLGGLLTM. Activated T-cells were selected by the cytokine secretion assay and expanded for 10 days. In 85% of donors 1.7 x106 (range 0.7 –4.5 x106; n=13) LMP-1 or LMP-2a specific CD8+ T-cell could be generated with an average purity of 83% as determined by tetramer staining. LMP1- and LMP2a-specific CD8+ T-cells were then expanded 3000 x in 14 d by the rapid expansion protocol and evaluated functionally for cytokine production and specific lysis. Both LMP-1 and LMP-2a specific CD8+ T-cells retained specific cytokine production if stimulated with peptide pulsed targets, efficiently lysed peptid pulsed targets. Surprisingly, if LMP-1 was presented endogenously by EBV positive targets or by targets cells transduced with LMP-1, no cytokine production or specific lysis was detected despite protein expression of LMP-1 in all targets. In contrast, IFN-γ production could be readily detected in LMP-2a-specific CD8+ T-cells after stimulation with target cells processing endogenously the LMP-2a antigen as well as specific lysis of EBV positive target cells. Furthermore, LMP2a specific CD8+ demostrated also specific lyse of Hodgkin-cells expressing the LMP2a (30:1 E/T ratio; 29,3%) where as LMP-1-specific CD8+ T-cells could not lyse HD-cells. In summary, LMP-1 and LMP-2a specific T-cells, although present at undectable levels in healthy donors, can be readily selected and expanded to up to 6x109 antigen-specific T-cells in less than 4 weeks starting from 1x108 PBMC. Based on this data, adoptive immunotherapy of relapsed EBV positive HD after allogeneic SCT should be preferentially performed with LMP-2a specific CD8+ T-cells rather than with LMP-1 specific CD8+ T-cells.

PKC beta II Expression in Lymphoid Malignancies.

Protein Kinase C (PKC) is an important enzyme family. Twelve different isozymes have been described, which are implicated in diverse cell responses. Recently, studies have begun to define isoform-specific functions of PKC, and PKC beta II seems to play an antiapoptotic role. PKC beta II expression was analyzed by immunohistochemistry in 119 tumor samples of patients with lymphoid malignancies. The median age of the group was 55 years (range 14–85 y). Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissue, using the labeled avidin-biotin immuno-peroxidase complex method on the Ventana Benchmark automated immunostainer (Ventana Medical Systems, S.A., Illkirch, France). The sections were incubated with primary antibody (polyclonal antibody cPKC ßII (C-1): sc-210, Santa Cruz Biotechnology, Inc.) for 32 min at 1/100 dilution. The intensity of cytoplasmic and membrane immunostaining was assessed semi-quantitatively by two pathologists independently. Cases were considered “negative” when <10% of cells were stained, and “positive” when ≥10% of tumor cells showed cytoplasmic or membrane staining. Diffuse or heterogeneous staining was also specified.Intense and diffuse staining was observed in all mantle cell lymphomas (13 cases, 100%), and chronic lymphocytic lymphomas (CCL) (6 cases, 100%). PKC beta II was positive too in most follicular lymphomas (10 cases out of 11, 91%), although the pattern and the intensity of the staining were variable in accordance with the degree of lymphomatous infiltration. Indeed, in the same lymph node, the follicular lymphomatous areas were constantly labelled, whereas residual germinal centers remained negative. A majority of angioimmunoblastic T-cell lymphomas, lymphoblastic T-cell lymphomas and Marginal Zone/MALT lymphomas were labelled with anti-PKC beta II antibody, but the pattern of expression was more heterogeneous in these subtypes. A minority of diffuse large B-cell lymphomas expressed PKC beta II (8 out of 22 cases, including 4 with weak staining). Nearly all plasma cell neoplasms were negative. Nevertheless, 2 cases out of 16 (13%) displayed a diffuse moderate staining. In peripheral T-cell lymphomas, staining for PKC beta II was less frequent, as well as in primary cutaneous B and T lymphomas and pseudo-lymphomas. None of the cases of Hodgkin's disease (13 cases) and anaplastic large cell lymphoma (5 cases) expressed PKC beta II. However, most of the numerous reactive cells were strongly stained.In conclusion, the intensity and the staining pattern of anti-PKC beta II antibody varied greatly according to the type lymphoid malignancy. These results highlight a significant correlation between PKC beta II expression and some particular type of lymphomas. In our series, the highest level of expression was found in mantle cell lymphomas and CLL.

SGN-30 (Anti-CD30 Monoclonal Antibody) Is Active and Well Tolerated in Patients with Refractory or Recurrent Systemic Anaplastic Large Cell Lymphoma.

SGN-30 is a chimeric mAb which recognizes the CD30 antigen found on tumor cells from patients (pts) with Hodgkin's disease (HD) and anaplastic large cell lymphoma (ALCL). In preclinical experiments, SGN-30 was shown to have antitumor activity in both in vitro and in vivo models of HD and ALCL. The results of a multi-dose phase I study showed minimal toxicity associated with doses of 2–12 mg/kg as six weekly IV infusions. One complete response (CR) was seen in the three non-Hodgkin's lymphoma patients (2 ALCL, 1 diffuse large B-cell lymphoma) accrued to the study and two patients demonstrated stable disease (SD) over time. A phase II multi-dose study was initiated to further evaluate the safety, antitumor activity and pharmacokinetics of six weekly IV infusions of 6 mg/kg of SGN-30 in pts with relapsed or refractory HD or systemic ALCL (sALCL). Five patients (2M, 3F) with ALCL have been enrolled, with a median age of 52 (range 33–75) and 3 median prior therapies (range 2–5). Multiple doses of SGN-30 have been well tolerated in all of the pts. Drug-related adverse events have been typically mild and consistent with mAb administration. No drug-related grade 3/4 events have been observed. Two patients have had objective responses with one patient achieving a CR. The patient's baseline CT scan showed a 5.1 x 2.0 cm chest wall mass in the lower left anterior lateral chest wall representing local lymphoma recurrence. After six doses of SGN-30 the mass disappeared completely with some edema in the chest wall but no obvious residual mass. The duration of response is pending at this time, and it will be presented in the meeting. Another patient experienced a partial response. The patient, who entered the study with constitutional symptoms and extensive cutaneous and nodal disease, had resolution of all skin lesions, significant reduction in adenopathy and improvement in constitutional symptoms after completion of her SGN-30 therapy. The patient progressed after discontinuation of therapy. Of the other three patients, one had SD and two progressed. These early results are promising and accrual to the trial continues.

Phase II Study of SGN-30 (Anti-CD30 Monoclonal Antibody) in Patients with Refractory or Recurrent Hodgkin's Disease.

SGN-30 is a chimeric monoclonal antibody which recognizes the CD30 antigen found on tumor cells from patients with Hodgkin's disease (HD) and anaplastic large cell lymphoma (ALCL). Preclinical studies with this agent have demonstrated anti-lymphoma effects in both in vitro cell line assays and in vivo murine model systems. The results of a multi-dose phase I study showed minimal toxicity associated with doses from 2 to 12 mg/kg administered as six weekly IV infusions over 120 minutes each. Of the 21 patients with Hodgkin's Disease accrued to the phase I study, four patients had stable disease (SD). A phase II multi-dose study is currently underway to further evaluate the safety, antitumor activity and pharmacokinetics of six weekly IV infusions of 6 mg/kg of SGN-30 in patients with relapsed or refractory HD or systemic ALCL (sALCL). Fifteen subjects (6M, 9F) with HD have been enrolled, with baseline data as follows: median age 34 (range 20–65), median number of prior therapies 3 (range 1–5), and 11 patients (73%) have disease which progressed after prior high-dose chemotherapy and stem cell transplant. Multiple doses of SGN-30 have been well tolerated in all subjects. Drug-related adverse events have been typically mild and consistent with monoclonal antibody administration. The most common drug-related adverse event has been fatigue. No grade 3/4 events have occurred. Twelve patients are currently evaluable for response, with 6 having had stabilization of their disease. Assessment of duration of disease stabilization and response is continuing. While the acceptable safety profile and frequency of stable disease following therapy in this heavily pretreated patient population are encouraging, a 12 mg/kg/dose regimen is now being tested in subsequent subjects to further explore the dose-response relationship of SGN-30 in Hodgkin's Disease. Further evaluation of this novel immunotherapy in additional HD patients is ongoing.

Differential Effects of Three Small Molecules Blocking Phosphatidylinositol-3 Kinase or AKT in Hodgkin Disease Cell Lines: Induction of Apoptosis and Cell Cycle Arrest.

Several types of tumor cells utilize the phosphatidylinositol 3 Kinase (PI3K)/AKT pathway to support their growth and survival, and blockade of this pathway has been shown to have an antiproliferative effect in these cells. The significance of this survival pathway in Hodgkin disease (HD), and in particular, the neoplastic Hodgkin and Reed-Sternberg (H/RS) of Hodgkin disease (HD) is unknown. We studied routinely processed tissue sections of 42 cases of primary HD (n=42, 35 classical HD and 7 nodular lymphocyte predominant) for the expression of the active phosphorylated form of AKT. We also studied AKT expression in a panel of 4 well-characterized HD-derived cell lines (HD-MyZ, HD-LM2, L-428, and KM-H2). 27 of 42 (64.3%) cases of primary HD sections expressed phospho AKT (23/35 classical and 4/7 nodular lymphocyte predominant). Cultured H/RS cells also showed constitutive phosphorylation of AKT on Ser473. CD30 ligand (CD30L), CD40L and RANKL, increased the phosphorylation of AKT and downstream molecules as early as 1 hour. The effect of 3 small molecule inhibitors of PI3K/AKT pathway (PI3K inhibitor LY294002; AKT inhibitors QLT0394 and QLT0395 from QLT Inc, Vancouver, Canada) on cell proliferation and survival of cultured H/RS cells was examined by the MTS assay. The PI3K inhibitor LY294002 showed antiproliferative activity in a time and dose dependent manner in all cell lines tested. This antiproliferative effect was primarily due to cell cycle arrest in G0/G1 phase, as determined by propidium iodide staining, and to a less extent due to induction of apoptosis, as determined by the Annexin-V binding assay. However, when the AKT inhibitors QLT0394 and QLT0395 were used, they primarily induced apoptosis even at nanomolar doses. Apoptosis was mediated by caspase 8 activation as determined by western blot and was partially reversed by the pancaspase inhibitor ZVAD-FMK. Both AKT inhibitors alteredf AKT phosphorylation within 24 hours, and reduced phosphorylation of downstream molecules, such as 4E-BP1. Moreover, the AKT inhibitor QLT0395 showed downregulation of MDM2 and cyclin D1 within 24 hours, and increased ERK1/2 phosphorylation with subsequent decrease of total ERK1/2 levels at the same time frame, but had no effect in BCL-2, cFLIP or Bax. Importantly, both AKT inhibitors (QLT0394 and QLT0395) maintained their killing activity even in the presene of CD30L, CD40L, and RANKL. These preclinical data suggest that small molecules hitting different targets within PI3K/AKT pathway may have different effects on proliferation and apoptosis and that blockade of this pathway may be of therapeutic value in the treatment of patients with HD.

The Geldanamycin Analogue Heat Shock Protein 90 (HSP90) Inhibitor 17-AAG in Lymphomas: Molecular Mechanisms of 17-AAG-Induced Cell Death in Hodgkin Disease and Non- Hodgkin Lymphoma Cell Lines.

Heat Shock Proteins (HSP) are stress-induced proteins that act as molecular chaperones in all benign and malignant cells. HSPs are important for maintaining the function of several client proteins involved in transcriptional regulation, signal transduction, and cell-cycle control. HSP90 has been recently identified as a potential therapeutic target in several types of tumors. However, the effect HSP90 inhibition in Hodgkin disease (HD) and non-Hodgkin lymphomas (NHL) remains unknown. The geldanamycin analogue 17-allylamino-17-demethoxy-geldanamycin (17-AAG) is a small molecule inhibitor of HSP90, which interferes with ATP-dependent chaperones activity, with subsequent degredation of client proteins by the proteasome. We studied the activity of 17-AAG in a panel of cell lines of HD (HD-MyZ, HD-LM2, L-428, KM-H2) and NHL (SUDHL1, and Jurkat) origin. HSP90 was expressed in all HD cell lines but not in normal peripheral blood lymphocytes of healthy donors. 17-AAG demonstrated a significant antiproliferative effect in a dose (01 micromolar - 5 micromolar) and time (24 hours to 72 hours) dependent manner in all cells lines, but was most prominant in the HD-LM2 cells. In all HD-derived cell lines, 17-AAG had more than 50% inhibitory effect over control cells. This effect was most prominant after 72 hours of incubation. Subsequently, we examined the molecular mechanisms responsible for this antiproliferative effect. Both cell cycle arrest and induction of apoptosis were observed. In the HD-LM2 cell line, 17-AAG predominantly induced a G0/G1 cell cycle phase arrest, while in the L-428 cell line there was a G2/M phase arrest (as determined by propidium iodide staining and flow-cytometric analysis). This effect was associated with alterations in MDM2 and cyclin D1 proteins. Cell cycle arrest was followed by apoptosis as determined by Annexin-V binding assay, which was predominantly mediated by caspase 9 activation. 17-AAG also reduced the cellular content of Bcl-2, cFLIP, and AKT, but had no significant effect on XIAP or cIAP2. Furthermore, 17-AAG decreased the total ERK1/2 which has been shown to play an important role in HRS cell survival. 17-AAG-induced apoptosis was partially reversed by the pancaspase inhibitor ZVAD-FMK. Preliminary data suggested that 17-AAG did not enhance the effect of doxoorubicin or gemcitabine chemotherapy. These data suggest a first proof-of-principle that inhibition of HSP90 might be of therapeutic value in HD and NHL.

The Anti-CD30 Monoclonal Antibody SGN-30 Inhibits Hodgkins Disease Growth and Sensitizes Cells to Established Chemotherapeutics.

SGN-30, a chimeric monoclonal antibody with apoptotic-signaling activity against CD30-positive malignancies, is currently in Phase II clinical evaluation against anaplastic large cell lymphoma and Hodgkin's disease (HD). Mechanisms underlying SGN-30's anti-tumor activity were evaluated using cDNA array analysis of SGN-30-treated L540 HD cells. Changes in expression of growth and apoptotic related genes including cyclin D2 and p21CIP1/WAF1 followed treatment of L540 and L540cy cells with SGN-30. Intra-cellular staining showed increases in both activated caspase-3 and p21CIP1/WAF1 in L540cy. These alterations coincided with increased Annexin V/PI staining. In addition to directly inducing apoptosis, SGN-30 mediated alterations in gene expression and could sensitize cells to standard chemotherapeutics. To evaluate this potential, L540cy cells were treated for 24 h with SGN-30 followed by exposure to a panel of chemotherapeutics commonly used in HD therapy and the combinations quantified by the Combination Effects method. Most of the chemotherapies examined were at least additive or better in combination with SGN-30, with bleomycin producing the strongest synergistic response. In vitro data were confirmed by the significantly increased efficacy of SGN-30 and bleomycin against established L540cy HD tumor xenografts in SCID mice. Results suggest that in addition to direct cell killing, SGN-30 binding to CD30 on HD tumor cells effects both growth arrest and drug sensitization by altering gene expression of cell cycle and apoptotic-related genes. Importantly, tumor cell sensitization to established chemotherapies, in particular DNA damaging agents used to treat HD, suggests that SGN-30 could improve the outcome of current drug-based therapies for treating HD.

Epstein Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes (CTL) Expressing an Anti-CD30 Chimeric T Cell Receptor (CTCR) for the Treatment of Hodgkin's Disease (HD).

HD may be a suitable target for immunotherapy, and in patients with EBV-associated HD, adoptive transfer of EBV-CTL has produced disease responses. An alternative target is the CD30 molecule, which is present on the malignant cells of almost all patients with HD. CD30 is a member of the TNF superfamily and monoclonal antibodies directed to this antigen are currently under investigation in patients with relapsed HD. An alternative way to target CD30 is by the construction of T cells expressing cTcR specific for the antigen. T lymphocytes engineered to express this cTcR can specifically kill CD30+ HD cell lines {Cancer Res , 1998; 58:1116}. However, these chimeric molecules connect the antigen-recognition properties of CD30 antibodies with the endodomain of CD3ζ, which is insufficient to fully activate resting T cells to proliferate and release cytokines. As a consequence chimeric T cells that express these endodomains divide infrequently, lose activity and have performed poorly in-vivo. Full T cell activation requires receptor engagement to be accompanied by a sequence of co-stimulatory stimuli. We have shown that EBV-CTL can fulfill this need, since the co-stimulatory signals delivered by EBV-infected B cells after native receptor engagement ensure full functionality when the CTL subsequently bind to tumor cells through their cTcR. We first evaluated whether EBV-CTL can be redirected to kill CD30+ HD cell lines and whether they retain their specificity and antigen repertoire. EBV-CTLs were prepared from 8 EBV+ healthy donors using weekly stimulation with irradiated autologous EBV-transformed lymphoblastoid cell lines (LCL) in the presence of IL-2 (50U/mL). CTL were transduced after the 3rd stimulation and further expanded with 3–4 weekly LCL/IL-2 stimulations. The expansion rate of the transduced CTL was similar to that of control EBV-CTL. Transduced CTL retained killing of their autologous LCL targets through their native receptor (64.4±16% at 20:1 E:T ratio), and became able to lyse CD30+ malignant lymphoma targets through their cTcR (e.g. HDLM-2=45.4±16% and Karpas-299=42.5±17%). Killing of CD30+ tumor cells was significantly inhibited by preincubation with an anti-CD30 blocking antibody (16.5±12%). Of potential concern, however, is that CD30 is expressed by activated normal T lymphocytes: expression was undetectable on resting T cells, but increased to 3–32% on day 4–7 after stimulation with LCL. Fortunately, expression dwindles to 3–6% by two weeks as an EBV-specific line emerges, suggesting that CD30 is expressed only in the early phases of T cell activation. As anticipated from these data, therefore, expression of a CD30 cTcR did not impair the antigenic repertoire of the EBV-CTL, which retained the same pattern of immunodominant MHC class I epitopes (detected by tetramer) as control cells. We also performed co-culture experiments to evaluate whether infusion of CTL-CD30 cTcR could cross-compromise the primary reactivation of other virus-specific CTL. Autologous EBV-CTLs engineered to express the CD30-cTcR were added to cultures of PBMC stimulated to reactivate cytomegalovirus- or adenovirus-specific CTL. In 4/4 donors, the percentage of CMV pp65+ T cells did not change, while generation of adenovirus-specific T cells (Hexon-tetramer+) was significantly reduced in only 1/3 donor. These data support the feasibility of using EBV-CTL bearing a cTcR for CD30 to treat both EBV+ and EBV− HD.

Induction of cell cycle arrest and apoptosis by the proteasome inhibitor PS-341 in Hodgkin disease cell lines is independent of inhibitor of nuclear factor-kappaB mutations or activation of the CD30, CD40, and RANK receptors.

The malignant Hodgkin and Reed-Sternberg cells of Hodgkin disease (HD) are known to constitutively express high levels of activated nuclear factor kappaB (NF-kappaB), which plays an important role in their survival. The proteasome inhibitor PS-341 has been recently shown to modulate tumor cell proliferation and survival by inhibiting NF-kappaB and modulating critical cellular regulatory proteins, but its activity in cells carrying IkappaBalpha gene mutations has not been reported previously. The activity of PS-341 in four well-characterized, HD-derived cell lines. Cell proliferation and apoptosis were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) and Annexin-V binding methods, respectively. Cell cycle analysis was determined by flow cytometry. Intracellular protein levels were determined by Western blot. PS-341 demonstrated a strong antiproliferative activity, which was irrespective of the status of mutations in IkappaBalpha and even the presence of CD30, CD40, or RANK receptor activation. This effect was attributable to the induction of apoptosis and cell cycle arrest at the G(2)-M phase. PS-341 not only inhibited nuclear localization of NF-kappaB but also activated the caspase cascade, increased p21 and Bax levels, and decreased Bcl-2 levels. Furthermore, PS-341 enhanced the effect of gemcitabine chemotherapy and potentiated the effect of tumor necrosis factor-related apoptosis-inducing ligand/APO2L and two agonistic antibodies to tumor necrosis factor-related apoptosis-inducing ligand death receptors R1 and R2. The in vitro activity of PS-341 against HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD.

Expression of CCL28 by Reed-Sternberg Cells Defines a Major Subtype of Classical Hodgkin's Disease with Frequent Infiltration of Eosinophils and/or Plasma Cells

Classical Hodgkin's disease (HD) is characterized by rare neoplastic Hodgkin and Reed-Sternberg (H-RS) cells within abundant reactive cellular backgrounds. In most cases, H-RS cells originate from the B-cell lineage, but their immunophenotypes are unusual. Here we newly found frequent expression of chemokine receptors CXCR6 and CCR10 and their respective ligands CXCL16 and CCL28 in HD-derived cell lines. CCR10 is known to be selectively expressed by plasma cells, whereas CCL28 attracts eosinophils via CCR3 and plasma cells via CCR10 and CCR3. Therefore, we examined their expression in HD tissues by immunohistochemistry. We found that H-RS cells in 15 of 19 cases were positive for CCL28. Among them, seven cases were also positive for CCR10, suggesting a potential autocrine effect. In situ hybridization confirmed the expression of CCL28 mRNA in H-RS cells. The CCL28 positivity in H-RS cells did not significantly correlate with that of LMP-1, CCL17, CCL22, or CCL11. However, it significantly correlated with the background accumulation of eosinophils, plasma cells, and CCR10+ cells. Thus, the production of CCL28 by H-RS cells may play a major role in tissue accumulation of eosinophils and/or plasma cells in classical HD. The frequent expression of CCR10 in H-RS cells themselves also supports their close relationship to plasma cells.

MEK/ERK pathway is aberrantly active in Hodgkin disease: a signaling pathway shared by CD30, CD40, and RANK that regulates cell proliferation and survival

AbstractThe mitogen-activated protein kinase (MAPK) (also called extracellular signal–regulated kinase [ERK]) pathway has been implicated in malignant transformation and in the regulation of cellular growth and proliferation of several tumor types, but its expression and function in Hodgkin disease (HD) are unknown. We report here that the active phosphorylated form of MAPK/ERK is aberrantly expressed in cultured and primary HD cells. Inhibition of the upstream MAPK kinase (also called MEK) by the small molecule UO126 inhibited the phosphorylation of ERK and demonstrated a dose- and time-dependent antiproliferative activity in HD cell lines. UO126 modulated the levels of several intracellular proteins including B-cell lymphoma protein 2 (Bcl-2), myeloid cell leukemia–1 (Mcl-1) and caspase 8 homolog FLICE-inhibitory protein (cFLIP), and induced G2M cell-cycle arrest or apoptosis. Furthermore, UO126 potentiated the activity of apoliprotein 2/tumor necrosis factor–related apoptosis-inducing ligand (APO2L/TRAIL) and chemotherapy-induced cell death. Activation of CD30, CD40, and receptor activator of nuclear kappaβ (RANK) receptors in HD cells by their respective ligands increased ERK phosphorylation above the basal level and promoted HD cell survival. UO126 inhibited basal and ligand-induced ERK phosphorylation, and inhibited ligand-induced cell survival of HD cell lines. These findings provide a proof-of-principle that inhibition of the MEK/ERK pathway may have therapeutic value in HD.

High-level expression ofBCL3differentiates t(2;5)(p23;q35)-positive anaplastic large cell lymphoma from Hodgkin disease

Anaplastic large cell lymphoma (ALCL) with t(2;5)(p23;q35) and Hodgkin disease (HD) share many cellular features, including expression of CD30. We compared gene expression profiles of 4 ALCL (Karpas 299, SU-DHL-1, DEL, SR-786) and 3 HD cell lines and found that BCL3, which encodes a nuclear protein belonging to the I kappa B family of inhibitors of nuclear factor-kappa B (NF-kappa B) transcriptional factors, was expressed at higher levels in ALCL than HD. Northern and Western blotting analyses confirmed the high-level expression of BCL3 in ALCL at both mRNA and protein levels. We established a real-time reverse transcriptase-mediated polymerase chain reaction assay to measure the BCL3 mRNA level and found a predominant level of BCL3 expression in t(2;5)(+) ALCL; the levels of cell lines and clinical materials were comparable to or higher than that of a B-cell chronic lymphocytic leukemia carrying t(14;19)(q32;q13). Southern blotting and fluorescence in situ hybridization disclosed that the BCL3 gene copies were amplified in SU-DHL-1, whereas Karpas 299 carried 4 BCL3 gene loci. The BCL3 gene contains 2 cytosine-guanine dinucleotide (CpG) islands, and the intragenic 3' CpG was entirely demethylated in SU-DHL-1 and DEL. In contrast to HD, in which NF-kappa B was constitutively activated, ALCL cells consistently showed (p50)(2) homodimer binding activity on electrophoretic mobility shift assay. It is suggested that the high-level nuclear Bcl-3 sequesters the (p50)(2) homodimer to the nucleus, which may account for the contradictory effect of CD30 stimulation on ALCL and HD. We propose that BCL3 is overexpressed by genetic and epigenetic modifications, potentially contributing to the development of t(2;5)(+) ALCL.

Comparison of Fascin Expression in Anaplastic Large Cell Lymphoma and Hodgkin Disease

Diagnostic difficulties sometimes arise in distinguishing anaplastic large cell lymphoma (ALCL) from Hodgkin disease (HD), especially the syncytial variant. Study of the biologic features of diagnostic Reed-Sternberg cells in HD, in search of specific markers for Reed-Sternberg cells, has suggested fascin as a relatively specific and sensitive marker. We studied the frequency of fascin expression in 30 ALCLs and 34 cases of classic HD, including 17 cases of the syncytial variant. Staining with CD30 and anaplastic lymphoma kinase (ALK)-1 also was performed in all cases. All ALCL and HD cases showed membranous and Golgi zone CD30 positivity. Fascin stained all HD cases but also stained 67% (20/30) of the ALCLs in a cytoplasmic pattern. Fascin positivity was observed in 59% (10/17) of T-cell ALCLs and 77% (10/13) of null-cell ALCLs; ALK-1–positive ALCLs, regardless of origin, were usually fascin-positive (91% [10/11]). In conclusion, fascin shows strong positivity in all cases of classic HD but also is positive in the majority of ALCLs, including ALK-1–positive and ALK-1–negative cases. Positive staining for fascin is not useful for distinguishing ALCL from HD. In some cases, fascin negativity may help rule out classic HD.