Hodgkin's Disease Cell Research Articles

Pharmacological Approach for Prevention of Bleomycin-Induced Lung Injury

Bleomycin is a chemotherapeutic medication commonly use in the treatment of Hodgkin's disease and germ cell tumors. Pulmonary fibrosis is major and dose limiting adverse effect of the drug. There is not currently any equal alternative approach or a medical agent for prevention of its pulmonary injury. We searched scientific databases in order to find investigational pharmacologic medicines in prevention of bleomycin-induced lung toxicity (BILI). Results revealed that some anti-inflammatory and antioxidant preparations such as statins, N-acetylcysteine (NAC), supplements (vitamin D3, L-arginine, selenium), and renin-angiotensin-aldosterone system (RASS) inhibitors might be effective in preclinical models of pulmonary toxicity mediated by bleomycin. However, we still need more in vivo studies and large human randomized clinical trials to confirm their benefits.

Polyclonal T Lymphocytes Transform into Malignant Cells by Retroviral Mediated Transfer of p21SNFT Gene

Background: p21SNFT (21 kDa small nuclear factor isolated from T cells) gene is known to be increasingly expressed in the cells of Hodgkin disease and Hodgkin cell lines. It was not investigated before if this gene had any effect on polyclonal T lymphocytes. For this purpose, we transduced this gene into polyclonal T lymphocytes and transplanted into mice. Materials and Methods: We used γ-retroviral vector to transduce this gene into polyclonal T lymphocytes, isolated from wild type black-6 mice. After confirmation of p21SNFT expression via marker gene eGFP (enhanced green fluorescent protein) and Western blot analysis, these cells were transplaned into immunocompromised Rag 1 deficient mice. The control group of mice was transplanted with γ-retroviral vector, carrying only marker gene (eGFP). The mice were followed up. The results showed that all 7 mice transplanted with p21SNFT-carrying polyclonal T lymphocytes developed massively enlarged mesenteric lymph nodes after 435 to 550 days of transplantation. Results: Histopathological analyses of these lymph nodes revealed the lymphoma, which resembled human peripheral T cell lymphoma type. Furthermore, immunophenotyping of tumor cells by FACS analysis revealed loss of T cell markers and expression of CD19 marker. From the control group, all five mice remained healthy and did not develop any tumor. Conclusion: The study concludes that γ-retroviral mediated transfer of p21SNFT transforms polyclonal T lymphocytes into malignant cells. Key words: γ-retroviral vector, Lymphoma, Malignant transformation, p21SNFT, T lymphocytes.

Correction: 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-κB Mutations or Activation of the CD30, CD40, and RANK Receptors

Correction: 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-κB Mutations or Activation of the CD30, CD40, and RANK Receptors

Dural lesions mimicking meningiomas: A pictorial essay

The purpose of this essay was to illustrate the radiological and pathological findings in a wide spectrum of dural lesions mimicking meningiomas. Familiarity with and knowledge of these findings will narrow the differential diagnosis and provide guidance for patient management. In this pictorial review, we describe the following entities: Solitary fibrous tumors, hemangiopericytoma, gliosarcoma, leiomyosarcoma, dural metastases, Hodgkin's disease, plasmocytoma, Rosai-Dorfman disease, neurosarcoidosis, melanocytic neoplasms and plasma cell granuloma.

Generating CTLs against the subdominant EBV LMP antigens by transient expression of an A20 inhibitor with EBV LMP proteins in human DCs

Epstein-Barr virus (EBV) infection leads to Hodgkin’s disease (HD) in some immunocompetent hosts. The malignant Reed-Sternberg cells of HD only express a limited array of subdominant EBV antigens to evade preexisting immune responses to EBV. The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed by HD and various EBV-associated malignancies, have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low in healthy EBV-infected hosts, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and the oncogenic potential. In the present study, we report that transitively expressing an inhibitor of A20, a key negative regulator of inflammatory signaling pathways, together with the LMP antigens (truncated LMP1 and full-length LMP2) greatly enhances maturation and cytokine production of human (h) monocyte-derived dendritic cells (DCs). As a consequence, LMP1/2-expressed, A20-silenced hDCs have an enhanced potency to prime LMP-specific T cell response. When the in vitro primed T cells are adoptively transferred into tumor-xenografted, severe combined immunodeficient (SCID) mice, some of the xenografted tumors approach complete regression. Thus, the study may provide an available resource of LMP-specific T cells for T cell immunotherapy.

Hodgkin Disease and the Role of the Immune System

Hodgkin disease (HD) is a malignancy of primarily B lymphocytes that has the unique ability to cause immunodeficiency, as well as provide immune evasion mechanisms to avoid self-destruction. In this review, the authors discuss Hodgkin disease, its association with Epstein-Barr virus (EBV), the immune deficiency caused by HD, and tumor immune evasion mechanisms. Specifically, the authors closely evaluate the roles of regulatory T cells in HD, cytotoxic T cells, cytokine and chemokine secretion, down-regulation of Fas ligand, and indoleamine 2,3-dioxygenase (IDO) secretion.

Ascorbate Promotes Epigenetic Activation of CD30 in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells have the ability to adapt to various culture conditions. Phenotypic and epigenetic changes brought about by the culture conditions can, however, have significant impacts on their use in research and in clinical applications. Here, we show that diploid hESCs start to express CD30, a biomarker for malignant cells in Hodgkin's disease and embryonal carcinoma cells, when cultured in knockout serum replacement (KOSR)-based medium, but not in fetal calf serum containing medium. We identify the commonly used medium additive, ascorbate, as the sole medium component in KOSR responsible for CD30 induction. Our data show that this epigenetic activation of CD30 expression in hESCs by ascorbate occurs through a dramatic loss of DNA methylation of a CpG island in the CD30 promoter. Analysis of the phenotype and transcriptome of hESCs that overexpress the CD30 signaling domain reveals that CD30 signaling leads to inhibition of apoptosis, enhanced single-cell growth, and transcriptome changes that are associated with cell signaling, lipid metabolism, and tissue development. Collectively, our data show that hESC culture media that contain ascorbate trigger CD30 expression through an epigenetic mechanism and that this provides a survival advantage and transcriptome changes that may help adapt hESCs to in vitro culture conditions.

Study of the morphological patterns and association of Epstein-Barr virus and human herpes virus 8 in acquired immunodeficiency deficiency syndrome-related reactive lymphadenopathy

Study of the morphological patterns of acquired immunodeficiency syndrome (AIDS)-related lymphadenopathy. We retrospectively selected cases of AIDS-related benign lymphadenopathy. Cases with lymphomas, frank granulomas and necrosis were excluded. We analyzed different morphological patterns and correlated these with immunophenotypic markers along with viral markers human herpesvirus 8-latency-associated nuclear antigen (HHV8-LANA), and Epstein-Barr virus-encoded ribonucleic acid (EBER) studies via in situ hybridization (EBER-ISH). We present the morphological patterns of 13 cases of human immunodeficiency virus (HIV)-reactive lymph nodes and their clinical, hematological, biochemical and radiological parameters with special emphasis on the presence or absence of viral markers, including HHV8 and EBV. Common patterns included follicular hyperplasia only (five cases), mixed pattern of follicular hyperplasia with burnt-out germinal centres (four cases), completely atretic follicle (two cases), folliculolysis (11 cases), dumbbell-shaped follicles (three each), progressive transformation of germinal centers (four cases), T-zone expansion (two cases), Reed Sternberg (RS) cells like immunoblasts (two cases), Castleman's-like features with lollipop-like follicles (three cases) and a spindle cell prominence (one case). CD8+ T-cells were predominant in 12 cases. CD8+ T-cells were prominent in germinal centers (eight cases). Plasmablasts were seen in four cases within the perigerminal center area. Immunohistochemistry for HHV8, i.e. HHV8-LANA were negative in all cases while EBER was detected in 11 cases in the centrocyte-like B cells. Two cases of multicentric Castleman's disease expressed EBER; however, they did not express HHV8. The wide spectrum of histological changes in HIV-associated lymphadenopathy requires recognition. The histological changes can mimic those of other infective lymphadenitis, follicular lymphoma, Castleman's disease, progressive transformation of germinal center, Hodgkin's disease and spindle cell neoplasms. Presence of EBV is common while HHV8 was not seen.

Potent antibody drug conjugates for cancer therapy

Significant progress has been made in the past few years in the area of antibody drug conjugates (ADCs) for the selective delivery of cytotoxic drugs to tumors. Early work in this field incorporated clinically approved drugs and mouse monoclonal antibodies (mAbs), which had modest activities, and were generally immunogenic. The results of these studies prompted investigation that led to the identity of several key parameters that influenced activity and tolerability. These included the antigen target, the use of non-immunogenic mAb carriers, the incorporation of highly potent drugs and novel conditionally stable linker technologies, and the specific methods used to attach drugs to mAbs. As a result of these investigations, new agents with pronounced clinical activities have been developed. These include SGN-35, an ADC directed against the CD30-positive malignancies such as Hodgkin's disease and anaplastic large cell lymphoma, and trastuzumab-DM1 which has shown activity in metastatic breast carcinoma. This review details many of the technological advancements, and provides examples of promising ADCs that are currently in clinical trials.

Increased luminol-enhanced chemiluminescence of blood monocytes and granulocytes in Hodgkin’s disease

The oxidative metabolic burst of blood monocytes and polymorphonuclear leukocytes (PMN) from 22 untreated patients with Hodgkin's disease (HD) and 18 healthy subjects were studied. Monocytes and PMN were enriched by density centrifugation and in vitro activated by zymosan. The oxidative metabolism was measured by luminol-enhanced chemiluminescence (CL). The CL of the patients' monocytes and PMN was higher than that of controls (P less than 0.01 and P less than 0.05, respectively). Patients with stage II-IV HD showed an increased blood monocyte CL as compared with stage I patients (P less than 0.05). Furthermore, patients with lymphocytic depletion or mixed cellularity subtype demonstrated an increased CL of PMN as compared with the remainder. Enhanced CL of phagocytes has been observed in chronic inflammatory disease and can be induced by various serum factors such as monokines and immune complexes. The present study demonstrates an increased CL of blood-borne phagocytic cells in untreated HD. Furthermore, CL of blood monocytes and PMN correlated to tumour burden and histologic subtype, respectively.

A 15-year-old girl with recurrent pneumonia

Hodgkin's disease (HD) represents a group of lymphomas with distinct clinical and histopathological features that account for approximately 5% of cancers in patients who are <15 years old and approximately 15% in patients who are 15-19 years old. Although the cause of HD is unknown, serologically confirmed infectious mononucleosis has been associated with an increased risk of HD, in addition to its known association with Burkitt's lymphoma. The Reed-Sternberg (RS) cell, a large and multinucleated cell with unique morphology, is the hallmark cell of HD. RS cells are clonal tumor cells that recently have been shown to be derived from B cells originating from germinal centers. Of four histological subtypes of HD, three have a good to excellent prognosis when recognized and treated early. We report a case of HD in a 15-year-old adolescent with a 14-month history of recurrent pneumonia. Open lung biopsy ultimately led to the diagnosis of HD. Although uncommon in this age group, the need to consider the possibility of neoplasm in the setting of recurrent respiratory infection is illustrated in this case. Early diagnosis and intervention may determine the prognosis of such neoplasms.

3D Nuclear Organization of Telomeres in Hodgkin and Reed-Sternberg Cells.

Telomerase activity is high in Hodgkin (H) and Reed-Sternberg (RS) cells and abundant telomerase RNA template (hTR) is identified in both by in situ hybridization. However, nothing is known about the three-dimensional (3D) structure of the telomeres in H and RS and the molecular events associated with the transition from the mononuclear H to the multinuclear diagnostic RS are still unclear. We analyzed the 3D structure of telomeres in interphase nuclei in the Hodgkin cell lines HDLM-2, L-428 and L-1236, where about 90 to 99% are mononuclear H and only 1 to 10% are multinuclear RS. We also analyzed the 3D structure of telomeres within 5 mm thin sections of three lymph node biopsies diagnostic for Hodgkin's disease (HD). The stereometric (3D) organization of telomeres was investigated in 30 H and 30 RS of each cell line and in 30 H and 30 RS of each lymph node biopsy specimen as previously described (Proc Natl Acad Sci USA , 102: 9613). Cellular localization of key-proteins of the telomere localized shelterin-complex (TRF1 and TRF2), of the mitotic spindle (centrin and gamma-tubulin), and of double-stranded DNA breaks (gamma-H2AX), was also analyzed. In all three cell lines the multinuclear RS showed overall significantly shorter and also significantly less telomeres in relation to the total nuclear mass when compared to their mononuclear H precursors. Visualization of their 3D telomeric structure revealed that this difference was due to partial or nearly total loss of telomeres within single nuclei of multinuclear RS; in particular, one or two nearly telomere free nuclei were often adjacent to one or two nuclei displaying several huge telomeric aggregates. TRF1 and TRF2 were mainly cytoplasmic in H and RS whereas gamma-H2AX accumulated in the nuclei of RS but not H. Multiple pairs of centrosomes not correlating with the number of nuclei, as well as high numbers of multi-polar spindles and incomplete spindles were identified in RS. Importantly, results analogous to those described in the HD cell lines were found in the HD lymph node biopsy specimen. In conclusion, our results suggest that multinuclear RS represent end stage tumour cells, where further nuclear division gets impossible due to sustained 3D telomere aggregation, shortening or loss. In particular, the number of nuclei within RS correlates closely with the 3D organization of telomeres, thus allowing for the first time a mechanistic understanding of the transition from H to RS cells. This process is initiated in H and advances to end stage telomere free “ghost” nuclei as observed in many RS. The shelterin complex appears to be disrupted and the mitotic cycle is profoundly disturbed.

HIV infection and cancer in the era of highly active antiretroviral therapy (Review)

The majority of cancers affecting HIV-infected subjects are those established as acquired immunodeficiency syndrome (AIDS)-defining: Kaposi's sarcoma (KS), non-Hodgkin's lymphoma (NHL), and invasive cervical cancer (ICC). However, other types of cancer, such as Hodgkin's disease (HD), anal cancer, lung cancer and testicular germ cell tumors appear to be more common among HIV-infected subjects compared to the general population. While not classified as AIDS-defining, these malignancies have been referred to as AIDS-associated malignancies. The mechanisms by which depressed immunity could increase the risk for cancer are unclear, except for in KS and most subtypes of NHL, where it is strictly associated with a low CD4 count. Although it remains unclear whether HIV-1 acts directly as an oncogenic agent, it may contribute to the development of malignancies through several mechanisms (e.g., infection by oncogenic viruses, impaired immune surveillance, imbalance between cellular proliferation and differentiation). Studies of the effect of highly active antiretroviral therapy (HAART) on the incidence and progression of HIV/AIDS-associated cancers provided contrasting data. While a significant decrease in the incidence of KS has been observed, HAART has not had a significant impact on NHL incidence, particularly systemic NHL, or on ICC, HD, anal cancers and other non-AIDS-defining cancers. Regardless of whether these cancers are directly related to HIV-induced immunodeficiency, treating cancer in HIV-infected patients remains a challenge because of drug interactions, compounded side effects, and the potential effect of chemotherapy on CD4 count and HIV-1 viral load. A better knowledge of viral mechanisms of immune evasion and manipulation will provide the basis for a better management and treatment of the malignancies associated with chronic viral infections.

Heterogeneity of protein kinase C β2 expression in lymphoid malignancies

Aims: Protein kinase C (PKC) β is an important regulator of lymphoid survival and its expression has been shown to be altered in lymphomas. The aim was to determine the expression of PKC β2 in various subtypes of lymphoproliferative diseases by immunohistochemistry.Methods and results: One hundred and forty archival samples representing various subtypes of lymphoproliferative diseases were analysed. Certain subtypes, such as mantle cell, lymphocytic or follicular lymphoma, were found to express PKC β2 in &gt; 90% of the samples. In follicular lymphomas, the follicular lymphomatous areas were constantly labelled, whereas residual germinal centres remained negative. In follicular hyperplasia, PKC β2+ cells were found in the mantle and marginal zones. Most angioimmunoblastic T‐cell lymphomas, lymphoblastic T‐cell lymphomas and marginal zone/mucosa‐associated lymphoid tissue (MALT) lymphomas were labelled with anti‐PKC βII antibody, but the pattern of expression was more heterogeneous in these subtypes. A minority of diffuse large B‐cell lymphomas were stained and most plasma cell malignancies were negative. None of the cases of Hodgkin's disease and anaplastic large cell lymphoma expressed PKC β2.Conclusions: PKC β2 expression varies significantly among lymphoproliferative diseases. In our series, the highest level of expression was found in mantle cell lymphomas and chronic lymphocytic lymphoma.

Improved Homing of Antigen-Specific T Cells to Hodgkin's Disease (HD) Tumor Cells by Forced Expression of CCR4 Receptor.

One crucial requirement for the success of any adoptive T cell transfer is that the effector T cells should migrate efficiently to the tumor site. Such an effect has been documented following adoptive transfer of Epstein-Barr virus specific cytotoxic T cells (EBV-CTLs). Antigenic stimulus from (normal and malignant) cells persistently infected with EBV led to expansion and sustained survival, and was also associated with activity against the EBV+ HD tumors. Since most HD patients have tumors that are EBV- but CD30+, we attempted to extend this approach by incorporating a CD30 chimeric receptor (CD30CAR) into the EBV-CTLs. Pre-clinical animal studies showed that CD30CAR+ EBV-CTLs readily migrated to EBV+/CD30+ tumors, but had limited capacity to localize to EBV−/CD30+ tumor cells. The likeliest explanation for this observation is that EBV- HD tumors produce the chemokine TARC, which attracts Th2 and regulatory T cells, but has little effect on EBV-CTLs, since these express low levels of the TARC receptor, CCR4. We hypothesized that forced expression of CCR4 by redirected EBV-CTLs would improve their homing to the EBV−/CD30+ HD cells. The full length of CCR4 receptor was cloned into the SFG retroviral vector and used to transduce both activated T cells and EBV-CTLs obtained from 6 and 4 healthy donors, respectively. Expression of CCR4 was 12±8% on activated T cells (mainly on CD4+ cells, 12±6%) and 4±5% on EBV-CTLs. After transduction with a CCR4, but not a control vector, expression of CCR4 increased to 53±24% (CD4+ 27±15% and CD8+ 17±9%) on activated T cells and 30±19% on EBV-CTLs. We then evaluated the capacity of control and transgenic T cells and EBV-CTLs to migrate in response to TARC, using a trans-well migration assay. Migration was tested against different CD30+ tumor lines producing TARC at low (Karpas wild type, <32 pg/ml) or high levels (Karpas engineered to produce TARC, HDLM-2 and L428, all TARC >2000pg/mL), measured by ELISA. The percent of cells migrating in the trans-well assay was significantly increased for CCR4 transgenic CD8+ selected T cells (54±11% with Karpas/TARC vs. 8±2% with media vs. 8±4% with Karpas-wt). Migration of control OKT3/28 blasts was less than 15% in all the conditions. Migration was significantly inhibited by the addition of antibody blocking TARC (<10%). An improved migration of CCR4+ CD8+ cells was observed also towards HDLM-2 (48±7%) and L428 (45±16%) as compared to control T cells (21±7% and 23±7%, respectively). Similarly, CCR4+ EBV-CTL showed improved migration towards Karpas/TARC (26±14%) as compared to Karpas/wt (12±8%) or media (10±7%). This genetic modification did not modify either the phenotype or the antigen specificity of EBV-CTLs, which retained the capacity to kill autologous EBV+ lymphoblastoid cells. We then determined if forced expression of CCR4 receptor was functional in vivo. To track homing of transgenic T cells, we transduced them with the FireFlyluciferase (FFLuc) vector and followed signal with a bioluminescent system (IVIS, Xenogen). Sublethally irradiated SCID mice were implanted s.c. with Karpas/wt on the left side and Karpas/TARC on the right side. When tumors were measurable, FFLuc+CCR4+ T cells were injected iv. By 24 hours post injection, signal was detectable only at the side of TARC secreting tumor cells, and signal persisted at 10 to 16 fold higher levels on the TARC+ side for >7 days. These data suggest that the migration of CARCD30 EBV-CTL to EBV−/CD30+ HD can be augmented by co-expressing the CCR4 receptor.

Efficacy of an Antagonistic Anti-CD40 Monoclonal Antibody, HCD122 (CHIR-12.12), in Preclinical Models of Human Non-Hodgkin's Lymphoma and Hodgkin's Disease.

Non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) account for about 9% of new cancer cases annually or 64,000 cases per year in the United States. Although the survival rate has significantly improved recently due to new combination therapy regimens, an unmet medical need remains for refractory or resistant patients. HCD122 is a fully human antagonistic anti-CD40 therapeutic monoclonal antibody (mAb) with a dual mechanism of action: blocking CD40 and CD40 ligand (CD40L) interactions and mediating antibody-dependent cellular cytotoxicity (ADCC). CD40 is expressed in all human B-cell malignancies, and the CD40/CD40L interaction is important for tumor cell proliferation and survival. Previously HCD122 was shown to potently inhibit CD40L-induced human B-cell and follicular NHL cell proliferation, mediate ADCC against CD40-positive human malignant B-cell lines and inhibit tumor growth in Burkitt's lymphoma and multiple myeloma xenograft models. In this study the antitumor activity of HCD122 was assessed in preclinical models of HD and other subtypes of human NHL, such as Mantle cell and Follicular lymphoma. CD40 was expressed in 5 of 7 established human HD and 11 of 12 NHL tumor cell lines tested, including Hs445, HDLM-2, KM-H2, L428, L1236, Jeko-1 and WSU-NHL. Using purified human NK cells as effector cells, HCD122 mediated potent ADCC against these cell lines in vitro with a picomolar EC50. When human macrophages were used as effector cells, HCD122 also induced antibody-dependent cellular phagocytosis (ADCP) against the NHL Daudi cell line and the HD cell line Hs445. The antitumor activity of HCD122 was further evaluated in vivo in EBV-negative NHL and HD xenograft models. When tested in a staged human Mantle cell lymphoma Jeko-1 s.c. xenograft model in which treatment was initiated when the mean tumor volume reached 100 mm3, HCD122 was highly efficacious and induced complete tumor regression in 70% (7/10) of treated animals when administered intraperitoneally at 1 mg/kg weekly for 4 weeks. In a staged human HD L428 s.c. xenograft model, which expresses CD20 as well as CD40, the antitumor activity of HCD122 was compared to rituximab. HCD122 was highly efficacious and induced a mean 74 % tumor growth inhibition (TGI) when administered at 0.1 mg/kg weekly for 3 weeks (p<0.001). At the same dose and schedule, rituximab achieved only 40% TGI (HCD122 vs. rituximab: p<0.001). These data combined with our previous studies in multiple myeloma and EBV-positive Burkitt's lymphoma models show that HCD122 is a potent anti-CD40 antibody with pronounced antitumor activity in both EBV-positive and EBV-negative malignant B cell preclinical models. HCD122 is currently in Phase I clinical trials in B-cell malignancies.

Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates

The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.

Loss of CD19 expression in B‐cell neoplasms

To investigate whether an antibody against an intracellular epitope can detect CD19 in routine biopsy specimens and thus to document in detail its expression in human lymphomas. A polyclonal antibody to the C terminus of CD19 was used to immunostain paraffin-embedded samples of normal and neoplastic lymphoid tissues. CD19 was widely expressed in normal B cells and in extramedullary plasma cells. It was found in most B-cell neoplasms, but expression in follicular lymphoma was weak (33/69) or negative (four cases). Similarly, CD19 expression in diffuse large B-cell lymphomas was weak (28/56) or negative (eight cases). In T-cell-rich B-cell lymphomas, CD19 was also weak (4/10) or negative (three cases). CD19 was often absent in post-transplant B lymphoproliferative disease, classical Hodgkin's disease and plasma cell neoplasms. An unexpected finding was the frequent absence of CD19 in the neoplastic cells in lymphocyte predominant Hodgkin's disease. CD19 can now be detected in routine biopsy specimens. In contrast to the classical pan-B marker CD20, CD19 is not always strongly expressed in B-cell neoplasms. Furthermore, the lymphocytic and histiocytic (L&H) cells of lymphocyte predominant Hodgkin's disease (which express most B-cell-associated markers) commonly lack CD19.

The Humanized Anti-CD40 Monoclonal Antibody SGN-40 Targets Hodgkin's Disease Cells through Multiple Mechanisms.

CD40 is a member of the TNF receptor superfamily expressed by multiple lineages within the hematopoietic system that carries out essential functions in the maturation of antigen-presenting cells, cross-priming of cytotoxic T cells, initiation of T cell-dependent antibody responses, and Ig class-switching. CD40 is widely expressed by lymphoid malignancies including B-cell precursor acute lymphoblastic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). SGN-40, a humanized version of the murine anti-CD40 monoclonal antibody S2C6, mediates antibody-dependent cellular cytotoxicity (ADCC) against both NHL and MM cells, directly induces apoptosis in NHL lines, and inhibits the proliferative response of MM cells toward autocrine IL-6 through down-modulation of the IL-6 receptor. Based on these activities, SGN-40 is currently in phase I clinical trials for relapsed and/or refractory MM and NHL. In this study, we examined the expression of CD40 on primary tumors isolated from 93 independent patients with HD. Seventy nine of these 93 cases (85%) demonstrated expression of CD40 by both the Reed-Sternberg cells as well as the infiltrating mononuclear cells. In a 1+ to 3+ scoring system, 35% of cases showed intense 3+ staining, 24% were positive (2+), and 26% were weakly positive (1+). Flow cytometry revealed that 8 of 9 HD cell lines expressed surface CD40, with receptor numbers ranging from 2,000 to > 370,000. SGN-40 induced CD40-positive HD cell lysis through ADCC in a dose-dependent manner, with IC50 values < 10 ng/mL. Fc-dependent effector function of SGN-40 was further exemplified by its ability to enhance macrophage mediated antibody-dependent cellular phagocytosis (ADCP). Specific ADCP against CD40-positive target cells was detected when SGN-40 was present at concentrations > 10 ng/mL. Furthermore, direct anti-proliferation signaling by SGN-40 in HD cells was also observed. In the L-1236 cell line, SGN-40 at doses ≥ 100 ng/ml reduced DNA synthesis to 25% of the untreated cells as measured by 3H-thymidine incorporation assays. Taken together, these data suggest that HD may be a suitable therapeutic target for SGN-40.

Coexpression of major histocompatibility complex class I and LMP1 in Epstein‐Barr virus‐positive Hodgkin's Lymphoma in a pediatric age group

The Epstein-Barr virus (EBV)-encoded latent membrane proteins (LMP1 and LMP2) are consistently expressed by malignant Hodgkin Reed-Sternberg (HRS) cells of EBV-associated Hodgkin's disease (HD). The EBV LMP1 has been implicated in tumorogenesis. Cytotoxic T lymphocyte (CTL) responses to both of these proteins have been shown in the blood of EBV-seropositive individuals, and in HD, the apparent failure of the CTL response to eliminate HRS cells expressing LMP1 in vivo has given rise to the suggestion that HD may be characterized by the presence of defects in antigen CTL-presentation or in CTL dysfunction. This study examined 28 cases seen at the Regional Children's Hospital Arturo Grullón in Santiago, the Dominican Republic, by immunohistochemistry. High level of expression of major histocompatibility complex (MHC) class I molecules in HRS cells of EBV-associated HD was recorded. These results suggest that deregulation of MHC class I molecules does not account for the apparent ability of EBV to infect HRS cells and to evade CTL protection. Therefore, the result from our work and similar studies will help to determine whether, or which, immunotherapy should be used in positive and negative cases of HD related to EBV.