C-myc Translocation Research Articles

Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in ApcMin/+ mice

BackgroundThe adenomatous polyposis coli (APC) protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E2 (PGE2) - PI-3 kinase pathways. Recent reports show that PGE2-induced phosphorylation of β-catenin by protein kinase A (PKA) increases nuclear translocation indicating two mechanisms of action of PGE2 on β-catenin homeostasis.FindingsTreatment of ApcMin/+ mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE2-induced β-catenin phosphorylation and c-Myc upregulation.ConclusionBased on our findings we suggest that PGE2 act through PKA to promote β-catenin nuclear translocation and tumor development in ApcMin/+ mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

The Role of Activation-Induced Cytidine Deaminase (AID/AICDA) in the Progression of Follicular Lymphoma

Abstract 5234Follicular lymphoma (FL), a entity with a cell origin in germinal center B lymphocytes, may develop into a progressive or transformed disease in some patients. This transformation is usually associated with acceleration of the clinical course. Transformed FLs generally retain the t(14;18) translocation, and it is believed that other genetic abnormalities are necessary in order for this transformation to occur. Secondary genetic events have been associated with this transformation such as c-myc amplification or mutation. Activation induced cytidine deaminase (AID/AICDA) is required for somatic hyper-mutation and class switch recombination of immunoglobulin gene, and also deeply involved in c-myc translocation of GCB-cell lymphoma. However, the role of AID in transformed FL has not been established. Here we tried to identify the significance of AID associated with c-myc in the progression of FL using mRNAs from tissue biopsy samples from patients with FL. The FLs we examined were divided into three groups: patients with grade 1–2 FL (n=15), patients with grade 3 FL who survived more than two years (n=14), and patients with rapidly progressive FL (RPFL) who died within two years after the start of treatment (n=6). AID and c-myc expression among those patients with FL were examined with RT-PCR and quantitative real-time PCR. Samples from the patients with grade 3 FL expressed relatively higher levels of the c-myc transcript than samples from the patients with grade 1–2 FL (p=.02) respectively. Additionally, nonsignificantly high levels of AID expression were observed in tissues from the patients with grade 3 FL. Interestingly, the samples taken from a patient with FL who died within two years after the start of treatment, showed either no or low expression of AID, despite expressing high levels of c-myc. These results suggested that overexpression of c-myc might be correlated with the biological transformation and clinical progression of FL; and also that AID might play a partial role in disease progression, and low expression of AID may be associated with the clinical features and outcome of patients with RPFL. In order to examine the role of AID expression in RPFL, the full-length AID gene was transfected into AID-negative and c-myc-positive cell lines established from patients with RPFL. AID-positive transfectants exhibited damping of cell proliferation proportional to the genetic level of AID expression, relative to the parental cells and the vector-only transfectants used as controls. Cell cycle analysis showed that the AID-positive transfectants had an increased cell population in G0/G1 phase and a reduced population in S phase, suggesting that G0/G1 arrest was responsible for the reduction in cell proliferation relative to the controls. These in vivo and in vitro findings suggest that AID may act as a negative regulator of cell survival in FL when sufficient c-myc is expressed. Switch-off or low expression of AID after c-myc amplification may be a potentially useful marker for prognostication of FL. Disclosures:No relevant conflicts of interest to declare.

Promoter and Exon 1 Hypermethylation of the Tumor Suppressor Gene PRDM1/Blimp-1 indicates Its Pathogenetic Role in EBV-Positive Burkitt Lymphoma,

Abstract 3471PRDM1/Blimp1, a lymphoma tumor suppressor and master regulator of plasma cell differentiation, is inactivated by genomic mutation and deletion in a subset of activated B-cell-type diffuse large B-cell lymphomas (DLBCL), underscoring the importance of terminal differentiation impairment in lymphoma pathogenesis. The role of PRDM1 inactivation in Burkitt lymphoma (BL) is currently not known. Two findings, however, may suggest possible PRDM1 involvement in BL pathogenesis. First, although PRDM1 protein is consistently absent in BL, about 40% of BL cases are positive for IRF4/MUM1, suggesting asynchronous expression of these two proteins. Secondly, it has been hypothesized that microRNA-127 overexpression in EBV+ BL may promote BL development through down-regulation of PRDM1. Sequence analysis of the coding region of PRDM1 in BL cell lines and primary tumors did not demonstrate any mutational changes. To investigate whether epigenetic inactivation of PRDM1 may play a pathogenetic role in BL, BL cell lines and primary cases were bisulfite sequenced to assess the methylation status of 41 CG dinucleotides in a 601 base-pair region spanning the distal promoter (DP) and a CG island that extends from the proximal promoter to the first exon of PRDM1. These include (i) 11 BL cell lines, 9 EBV+ (4 latency I, 2 Wp-restricted, and 3 with latency III drift) and 2 EBV-; (ii) 7 EBV+ lymphoblastoid cell line (LCL); (iii) 62 primary BL cases, formalin-fixed, paraffin-embedded tissues (FFPE); (iv) 4 B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCL-U), FFPE. Naïve, germinal center (GC) and memory B cells sorted by flow cytometry were also analyzed as controls. Hypermethylation in PRDM1 promoter and exon 1 was seen in all EBV+ BL cell lines except one with latency III, but not in EBV- BL lines. None of the LCL demonstrated PRDM1 hypermethylation, implying that EBV is unlikely to mediate PRDM1 hypermethylation directly. Methylation status of PRDM1 was successfully determined in 39 BLs (30 sporadic, 9 HIV-related) and 5 BCL-Us (4 sporadic, 1 HIV-related). Hypermethylation was seen in 11 BLs and 1 BCL-U (28.6% of total). All the methylated cases were EBV(+) (p=0.004). Overall, 12 of 28 (43%) EBV(+) BL and BCL-U cases exhibited hypermethylation. PRDM1 hypermethaylation was independent of BL subtype and MUM1/IRF4 expression status. To determine if PRDM1 hypermethylation can potentially repress PRDM1 transcription, BL cell lines Ramos (PRDM1 unmethylated) and Mutu1 (PRDM1 hypermethylated) are treated with IL-21 (50ng/mL), a cytokine that can mediate terminal differentiation by inducing PRDM1, for 2 to 5 days. While PRDM1 is induced by IL-21 in Ramos, levels of PRDM1 are not significantly increased in Mutu1, even though STAT3, a downstream mediator of IL-21, is activated in the latter. This finding suggests that PRDM1 hypermethylation has the potential to repress PRDM1 transcription in the presence of PRDM1-inducing signals. This function, however, may be pathogenetically more relevant in a BL precursor cell than in the final BL tumor cell. EBV+ BL is thought to derive from an EBV-infected late germinal center (GC) B cell, which harbors c-myc translocation and begins, though abortively, memory B cell differentiation and adopts an EBV latency I form. BL consistently express BCL6, a known PRDM1 transcription repressor, and harbor very low levels of PRDM1 mRNA. This suggests that high BCL6 expression, rather than PRDM1 hypermethylation, may be the primary determinant of its repressed transcription seen in BL. Indeed, PRDM1 mRNA expression is 25 to 1200 fold higher in LCLs which harbor very low levels of BCL. Furthermore, reducing expression of MTA3, which forms a complex with BCL6 and mediates PRDM1 transcription repression, in Daudi increases PRDM1 transcript levels. Therefore, we hypothesize that PRDM1 hypermethylation in BL more likely represents a memory of an epigenetic event that functions in the earlier stage of BL pathogenesis. PRDM1 hypermethylation may function to repress its transcription in a BL precursor cell as it is exposed to inducing signals, e.g. IL-21, during GC transit. Our study expands the spectrum of B cell lymphomas in which PRDM1 plays a tumor suppressor role, and supports the importance of impairment of terminal differentiation in the pathogenesis of a subset of aggressive B cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

5-Aza-2′-deoxycytidine Activates Iron Uptake and Heme Biosynthesis by Increasing c-Myc Nuclear Localization and Binding to the E-boxes of Transferrin Receptor 1 (TfR1) and Ferrochelatase (Fech) Genes

The hypomethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) and its derivatives have been successfully used for the treatment of myelodysplastic syndromes, and they frequently improve the anemia that usually accompanies these disorders. However, the molecular mechanisms underlying this action remain poorly understood. In this study, we used two erythroid models, murine erythroid leukemia cells and erythroid burst-forming unit-derived erythroblasts, to show that 5-aza-CdR induced erythroid differentiation and increased the expression of transferrin receptor 1 (TfR1) and ferrochelatase (Fech), thereby increasing iron uptake and heme biosynthesis. We have identified new regulatory E-boxes that lie outside of CpG islands in the TfR1 and Fech promoters, and the methylation status of these sites can be altered by 5-aza-CdR treatment. This in turn altered the binding of the transcription factor c-Myc to these promoter elements. Furthermore, 5-aza-CdR promoted the nuclear translocation of c-Myc and its binding to Max to form functional complexes. The coordinated actions of 5-aza-CdR on the methylation status of the target genes and in stimulating the nuclear translocation of c-Myc provide new molecular insights into the regulation of E-boxes and explain, at least in part, the increased erythroid response to 5-aza-CdR treatment.

The company malaria keeps

Co-infection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) are implicated in the cause of endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in equatorial Africa. Although the causal association between EBV and eBL has been established, P. falciparum malaria's role is not as clearly defined. This review focuses on how malaria may disrupt EBV persistence and immunity. Two mutually compatible theories have been proposed. One suggests that P. falciparum malaria induces polyclonal B-cell expansion and lytic EBV reactivation, leading to the expansion of latently infected B cells and the likelihood of a c-myc translocation, a hallmark of Burkitt lymphoma tumors. The other advocates that EBV-specific T-cell immunity is impaired during P. falciparum malaria co-infection, either as a cause or consequence of enhanced EBV replication, leading to loss of viral control. Advancements in our ability to query the complexity of human responses to infectious diseases have stimulated interest in eBL pathogenesis. EBV is necessary but not sufficient to cause eBL. A more dynamic model encompasses incremental contributions from both chronic and acute P. falciparum malaria leading to alterations in EBV persistence and EBV-specific immunity that culminate in eBL. A better understanding of how P. falciparum malaria modifies EBV infections in children may allow us to anticipate reductions in eBL incidence coinciding with malaria control programs.

Epstein-Barr Virus-Induced Gene 3 (EBI3): A Novel Diagnosis Marker in Burkitt Lymphoma and Diffuse Large B-Cell Lymphoma

The distinction between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), two types of mature aggressive B-cell lymphomas that require distinct treatments, can be difficult because of forms showing features intermediate between DLBCL and BL (here called BL/DLBCL). They can be discriminated by the presence of c-myc translocations characteristic of BL. However, these are not exclusive of BL and when present in DLBCL are associated with lower survival. In this study, we show that Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed among BL and DLBCL. Analysis of gene expression data from 502 cases of aggressive mature B-cell lymphomas available on Gene Expression Omnibus and immunohistochemical analysis of 184 cases of BL, BL/DLBCL or DLBCL, showed that EBI3 was not expressed in EBV-positive or -negative BL cases, whereas it was expressed by over 30% of tumoral cells in nearly 80% of DLBCL cases, independently of their subtypes. In addition, we show that c-myc overexpression represses EBI3 expression, and that DLBCL or BL/DLBCL cases with c-myc translocations have lower expression of EBI3. Thus, EBI3 immunohistochemistry could be useful to discriminate BL from DLBCL, and to identify cases of BL/DLBCL or DLBCL with potential c-myc translocations.

Biological feature validation of estimated gene interaction networks from microarray data: a case study on MYC in lymphomas

Gene expression is a dynamic process where thousands of components interact dynamically in a complex way. A major goal in systems biology/medicine is to reconstruct the network of components from microarray data. Here, we address two key aspects of network reconstruction: (i) ergodicity supports the interpretation of the measured data as time averages and (ii) confounding is an important aspect of network reconstruction. To elucidate these aspects, we explore a data set of 214 lymphoma patients with translocated or normal MYC gene. MYC (c-Myc) translocations to immunoglobulin heavy-chain (IGH@) or light-chain (IGK@, IGL@) loci lead to c-Myc overexpression and are widely believed to be the crucial initiating oncogenic events. There is a rich body of knowledge on the biological implications of the different translocations. In the context of these data, the article reflects the relationship between the biological knowledge and the results of formal statistical estimates of gene interaction networks. The article identifies key steps to provide a trustworthy biological feature validation: (i) analysing a medium-sized network as a subnet of a more extensive environment to avoid bias by confounding, (ii) the use of external data to demonstrate the stability and reproducibility of the derived structures, (iii) a systematic literature review on the relevant issue, (iv) use of structured knowledge from databases to support the derived findings and (v) a strategy for biological experiments derived from the findings in steps (i-iv).

Rapamycin Reverses Splenomegaly and Inhibits Tumor Development in a Transgenic Model of Epstein-Barr Virus–Related Burkitt's Lymphoma

Epstein-Barr virus (EBV) infection and latency has been associated with malignancies, including nasopharyngeal carcinoma and Burkitt's lymphoma. EBV encoded latent membrane protein 2A (LMP2A) is expressed in most EBV-associated malignancies and as such provides a therapeutic target. Burkitt's lymphoma is a hematopoietic cancer associated with the translocation of c-MYC to one of the immunoglobulin gene promoters leading to abnormally high expression of MYC and development of lymphoma. Our laboratory has developed a murine model of EBV-associated Burkitt's lymphoma by crossing LMP2A transgenic mice with MYC transgenic mice. Since LMP2A has been shown to activate the PI3K/Akt/mTOR pathway, we tested the therapeutic efficacy of mTOR inhibitor rapamycin on the tumors and splenomegaly in these double transgenic mice (Tg6/λ-MYC). We found that rapamycin reversed splenomegaly in Tg6/λ-MYC mice prior to tumor formation by targeting B cells. In a tumor transfer model, we also found that rapamycin significantly decreased tumor growth, splenomegaly, and metastasis of tumor cells in the bone marrow of tumor recipients. Our data show that rapamycin may be a valuable candidate for the development of a treatment modality for EBV-positive lymphomas, such as Burkitt's lymphoma, and more importantly, provides a basis to develop inhibitors that specifically target viral gene function in tumor cells that depend on LMP2A signaling for survival and/or growth.

Modulation of Bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase Cα

Protein kinase Cα (PKCα) is a classical PKC isoform whose involvement in cell death is not completely understood. Bax, a major member of the Bcl-2 family, is required for apoptotic cell death and regulation of Bax translocation and insertion into the outer mitochondrial membrane is crucial for regulation of the apoptotic process. Here we show that PKCα increases the translocation and insertion of Bax c-myc (an active form of Bax) into the outer membrane of yeast mitochondria. This is associated with an increase in cytochrome c (cyt c) release, reactive oxygen species production (ROS), mitochondrial network fragmentation and cell death. This cell death process is regulated, since it correlates with an increase in autophagy but not with plasma membrane permeabilization. The observed increase in Bax c-myc translocation and insertion by PKCα is not due to Bax c-myc phosphorylation, and the higher cell death observed is independent of the PKCα kinase activity. PKCα may therefore have functions other than its kinase activity that aid in Bax c-myc translocation and insertion into mitochondria. Together, these results give a mechanistic insight on apoptosis regulation by PKCα through regulation of Bax insertion into mitochondria.

TNFAIP3 (A20) Genetic Alterations In EBV Associated AIDS Related Lymphomas

AbstractAbstract 802 Introduction:AIDS related lymphomas (ARL) are a heterogeneous group of lymphoproliferative disorders that are frequently associated with Epstein Barr virus (EBV) infection. EBV expresses latent viral oncoproteins that constitutively activate the transcription factor NF-κB, a potent inducer of genes involved in B cell survival and proliferation (Keller SA et al, Blood 2006). Lymphomas that are not associated with EBV can also display increased NF-κB activity and recent reports have described mutations in regulators of NF-κB in subsets of B cell lymphomas. One of the frequently mutated regulatory genes is TNFAIP3, which encodes A20, an ubiquitin modifying enzyme involved in the termination of NF-κB signaling. Mutations resulting in the inactivation of A20 have been found in a significant proportion of marginal zone lymphoma (Novak U et al, Blood 2009), classical Hodgkin lymphoma, primary mediastinal B cell lymphoma (Schmitz R et al, J Exp Med 2009), and diffuse large B cell lymphoma (Compagno M et al, Nature 2009). In ARL the incidence of alterations in A20 and the relationship with EBV infection has not been described. Materials and Methods:We evaluated archival formalin fixed paraffin embedded tissue samples of ARL for genetic alterations in A20. Tissue was collected through an international collaboration between Weill Cornell Medical College in New York, USA and Siena University in Siena, Italy. A tissue microarray with 46 cases of ARL was prepared and characterization of lymphoma subtype and EBV viral status were determined by immunohistochemistry and in situ hybridization for Epstein-Barr encoded RNA. Fluorescent in situ hybridization (FISH) was used to evaluate for genomic deletions in A20, and translocations of cMYC, BCL-2 and BCL-6. Direct sequencing of the coding region and splice sites of A20 was performed to evaluate for additional genetic alterations. Immunohistochemistry was used to evaluate for the presence of A20 protein. Results:Fluorescent in situ hybridization revealed A20 monoallelic or biallelic deletion in 6 of 25 cases (24%). A20 point mutations were found in 3 of 23 cases (13%). Nonsense mutations coding for a premature stop codon in exon 2 were seen in 2 cases. The third case was found to have a missense mutation in exon 7 resulting in an amino acid change. Two of the 3 cases with an A20 point mutation had A20 deletion in the complementary allele indicating biallelic alteration of the A20 gene. Immunohistochemistry for A20 was performed and is reported for the first time in this abstract. Absence of A20 protein was demonstrated in 4 of 33 samples (12%). Included among the cases negative for A20 on immunohistochemistry is the single case with biallelic A20 deletion demonstrated by FISH. In total 10 of 39 (26%) cases with adequate sample for evaluation were determined to have inactivation of A20 by FISH, sequencing, immunohistochemistry, or a combination. A20 inactivation was seen among all histologic subtypes of ARL including Burkitt lymphoma (n=2), diffuse large B cell lymphoma of the germinal center B cell (n=2) and non-germinal center B cell (n=2), plasmablastic lymphoma (n=3) and B cell lymphoma, unclassifiable, intermediate between BL and diffuse large B cell lymphoma (n=1). Interestingly, the incidence of EBV infection was higher in cases with A20 inactivation than in those with intact A20. EBV was present in 6/10 cases with A20 alteration (60%) vs. 8/29 cases with intact A20 (28%). The EBV latent viral protein LMP-1, which activates NF-κB, was not expressed in cases with A20 alteration. Conclusions:This is the first report to demonstrate A20 inactivation in EBV-associated lymphoma. A20 molecular analysis has been previously reported in Hodgkin Lymphoma (HL) where A20 inactivation and EBV infection were found to be almost mutually exclusive (Schmitz R et al, J Exp Med 2009). The EBV gene expression pattern differs in HL and ARL. In HL EBV expresses the viral oncoprotein LMP-1, which leads to constitutive activation of NF-κB. In ARL viral gene expression is more heterogeneous and in this cohort of ARL, LMP-1 was not expressed in any of the cases with EBV infection and A20 loss. Our data indicate that A20 may represent a tumor suppressor gene in a significant subset of ARL and that A20 inactivation may be associated with positive EBV status. In EBV related lymphoma inactivation of A20 may be an alternative mechanism of NF-κB upregulation in the absence of LMP-1. Disclosures:No relevant conflicts of interest to declare.

Prognostic Impact of Germinal Center (GC)/ Activated B-Cell (ABC) Classification Analysed by Immunochemistry, FISH Analysis and GEP, In Relapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL): The Bio-CORAL Study

AbstractAbstract 993 Purpose:Cell of origin (COO) has a major prognostic impact in DLBCL. However this impact has never been analysed in the context of relapsed/refractory DLBCL. The purpose of this study was to evaluate the prognostic value of COO immunohistochemical markers and BCL2, BCL6 and c-MYC translocations in a well-defined cohort of patients included in the CORAL trial1. Methods:Enrolled patients had CD20+ DLBCL in first relapse or refractory after upfront therapy. Treatment was randomized to either R-ICE (rituximab, ifosfamide, etoposide, carboplatin) or R-DHAP (rituximab, dexamethasone, aracytine, cisplatin). Responding patients had high-dose chemotherapy and ASCT. Paraffin-embedded tumor samples of 249 patients were analysed by immunochemistry for CD10/MME, BCL6, MUM1/IRF4, FOXP1, BCL2 expression and for BCL2, BCL6 and c-MYC breakpoints by FISH, and were categorized according to different COO algorythms2-5. Primary biopsy at diagnosis and relapse biopsy were available for 189 and 147 patients respectively, including 87 matched pairs. Results:With a median follow-up at 27 months, there was no significant difference between R-ICE and R-DHAP for 2-year progression-free survival (PFS) (56.7%+/−5% vs 60%+/−5%, p=0.24) or overall survival (OS) (46%+/−5% vs 60%+/−5%, p=0.24). We confirmed that early relapse, prior rituximab exposure and secondary aaIPI had adverse prognostic value. In univariate analysis, absence of bcl6 expression, non-GC phenotype2, ABC phenotype4, at diagnosis were associated with an unfavourable PFS (p=0.038, p=0.0044, p=0.044, respectively), whereas only non-GC phenotype2 was associated to a worse OS (p=0.027). At relapse, expression of Foxp1 and bcl2, c-MYC breakpoint, ABC phenotype4, immunoFISH index5 was associated with an unfavourable PFS (p=0.02, p=0.027, p=0.03, p=0.032, p=0.02, respectively), and c-MYC breakpoint was associated with a worse OS (p=0.01). Tumor immunophenotype and FISH analysis at diagnosis and at relapse were statistically concordant in matched pairs (Wilcoxon's test between 0.42 and 1). In the whole group, only c-MYC breakpoint present in 13% of the patients was correlated with worse PFS (p= 0.02) and OS (p =0.04). Statistical interaction were found between GC/non-GC classification2 and the treatment R-ICE vs R-DHAP. Patients with GC B-cell like DLBCL subtype treated with R-DHAP have a better 2-year PFS (64% +/−7%) than those with GC B-cell like DLBCL treated with R-ICE (42% +/−7%), and the patients with ABC B-cell like DLBCL treated either with R-DHAP (35%+/−7%) or R-ICE (45%+/−7%) (p= 0.04). In multivariate analysis, only GC/non-GC phenotype2 interaction with the treatment, FOXP1 expression, prior rituximab exposure, secondary aaIIPI, time to relapse were statistically significant. Preliminary results of gene expression profiling seem to confirm these data and are currently under validation. Conclusion:COO remains a major factor in relapsed patients with a better response to R-DHAP salvage chemotherapy in GC-B DLBCL, underlying the importance of molecular DLBCL subtyping. Treatment of ABC subtype is still unsatisfactory.Gisselbrecht C, et J Clin Oncol. 2010.Hans CP, et al.Blood. 2004;103:275-282.Muris JJ, et al. J Pathol. 2006;208:714-723.Nyman H, et al. Mod Pathol. 2009;22:1094-1101.Copie-Bergman C, et al. J Clin Oncol. 2009;27:5573-5579. Disclosures:Schmitz:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gisselbrecht:Roche: Research Funding.

Establishment of An HS23 Stromal Cell-Dependent Myeloma Cell Line: Fibronectin and IL-6 Are Critical for Survival

AbstractAbstract 2949Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells triggers cytokine-mediated tumor cell growth, survival, and drug resistance. In particular, integrin a4b1, very late antigen 4 (VLA-4)-mediated fibronectin adhesion confers a survival advantage to myeloma cells. One of the problems in treating patients with MM is that it is very hard to eliminate residual myeloma cells, even following high-dose chemotherapy followed by auto-stem cell transplantation. Importantly, cell adhesion-mediated drug resistance (CAM-DR) must be overcome in order to eliminate the minimal residual disease of MM. Here we characterized a multiple myeloma cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first two months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and subsequently, they continued to proliferate on HS23 stromal cells but not on HS27A stromal cells (both stromal cells were established by Torok-Storb B, Blood 1995). The phenotype of the established MSG1 cell line was: CD138+, CD38++, CD19−, CD56−, VLA-4+, VEGFR1+, and VEGFR2+. Furthermore, immunohistochemical staining demonstrated expression of IgA and λ chain in the cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities, basically hypertriploidy including the deletion of chromosome 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured on fibronectin-coated plates with the supernatant of HS23 cells or RPMI1640 medium supplemented with 10% FBS (control medium) containing IL-6 (10 ng/ml). Notably, MSG1 could not survive in control medium containing IL-6 or in HS23 supernatant unless bound to fibronectin, which was also expressed on HS23 and HS27A cells. IL-6 and VEGF production were detected in the supernatants of both HS23 and HS27A stromal cells (36.8±4.5 pg/ml and 131±5.8 pg/ml; 13.2±1.9 pg/ml and 16664±418 pg/ml, respectively). Next, we analyzed the effect of tocilizumab, an anti-IL-6R antibody, and bevacizumab, an anti-VEGF antibody on MSG1 survival. Tocilizumab (50 μ g/ml) inhibited MSG1 survival when cultured on fibronectin-coated plates in control medium containing IL-6 (10 ng/ml), and tocilizumab (10 μ g/ml) inhibited MSG1 survival when cultured on HS23 stromal cells. However, bevacizumab (500 μ g/ml) did not show such inhibition. Therefore, MSG1 survival depends on HS23 stromal cells: in other words, it depends on binding to fibronectin and IL-6. If these factors induced CAM-DR in myeloma cells, MSG1 may be a unique myeloma cell line that will be useful for analysis of CAM-DR, and tocilizumab might be a useful drug for treatment of MM. Furthermore, since MSG1 could survive on irradiated HS27A cells, and since HS23 and HS27A express similar adhesion molecules (Torok-Storb B et al., Blood 1995), these data suggest that HS27A might secrete factors that are detrimental to MSG1 survival. The identification of such an inhibitory factors could be of interest in terms of the regulation of myeloma proliferation. Disclosures:No relevant conflicts of interest to declare.

Establishment of a Novel Cell Line of Plasmablastic Lymphoma with Loss of p16 Tumor Suppressor Protein and Overexpression of MDR

AbstractAbstract 4451Plasmablastic lymphoma is characterized by diffuse proliferation of large blastoid cells with immunophenotype of plasma cells. It frequently presents as a mass in the oral cavity, but it may also be encountered in other extranodal sites. It is clinically aggressive with a median survival of 6 months. Immunodeficiency, mainly by HIV, is the major risk factor for this disease, however, in some Asian countries like Japan and Korea, more numbers of HIV-negative cases of plasmablastic lymphoma has been reported, with fewer Epstein Birr Virus (EBV)-associated cases. Thus, it needs to be further determined whether any ethnic differences in the occurrence of this disease exists.We have experienced a rare case of plasmablastic lymphoma of the uterus in an otherwise healthy young Japanese female. Although treated with multiple chemotherapy regimens such as CHOP, hyper CVAD and VAD, the patient died of the disease within three months after the diagnosis. The clinical course of the disease was unique with de novo onset in the uterus, progressing to systemic lymph node swelling. Immunophenotype of the tumor was positive for CD138, VS38c and EMA but negative for CD20 and EBER. Karyotyping of the tumor showed two novel chromosome translocations, t(4;7)(q35;q22) and t(9;13)(p22;q22), either as a single translocation of t(9;13) or both. Southern blot analysis of the tumor was negative for EBV or c-myc translocation, which has recently been reported to have causal relationship with this type of lymphoma. Written informed consent was obtained from the patient and the family, and tissue sample obtained by lymph node biopsy was used for further analysis.The lymph node specimen was transplanted subcutaneously in immunodeficient NOG mice and was stably transplantable up to over five passages. The tumor was also transplantable subcutaneously to nude mice. We also succeeded in maintaining these newly established plasmablastic lymphoma cell lines in vitro. Two cell lines, one with a single translocation of t(9;13) and another with double translocation of t(4;7) and t(9;13) were established. Both cell lines were identical to the original patient's tumor in terms of immunophenotype.Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) was performed to identify the precise chromosomal breakpoints in these translocations. FISH analysis using approximately 50 probes identified consistent result with CGH analysis, which lead to the identification of lack of tumor suppressor p16 protein expression (t(9;13)) and upregulation of multidrug resistant protein (MDR) (t(4;7)).The expression of cell surface MDR and its function were also confirmed using flow cytometry and dye efflux method. Comparison among the established cell lines with or without the t(4;7) translocation confirmed increased resistance to chemotherapeutic drugs such as adriamycin and cisplatinum in t(4;7) positive, MDR overexpressing cell line. This result is in accordance with the clinical course of the patient, where adriamycin was initially the key drug, but lost its responsiveness with disease progression.We believe that this is the first report of the establishment of cell line for plasmablastic lymphoma that has identified genetic lesions involved in the pathogenesis of the disease. These newly established cell lines and experimental animal models of plasmablastic lymphoma will be a useful tool to understand the pathogenesis of the disease including development of the typical plasmablastic feature of this type of lymphoma, and to identify novel effective treatment strategy for this highly aggressive lymphoma. Disclosures:No relevant conflicts of interest to declare.

TfR1 and Ferrochelatase Expression During Erythroid Differentiation Induced by 5-Aza-Cdr In Both MEL Cells and BFU-E-Derived Erythroblasts Correlates with c-Myc Function

AbstractAbstract 4248During erythroid differentiation, heme biosynthesis increases dramatically and this is accompanied by a marked increase in transferrin receptor 1 (TfR1) expression and iron uptake from transferrin. It has long been known that 5-aza-2′deoxycytidine (5-aza-CdR), a demethylating agent, is able to induce murine erythroid leukemia (MEL) cell differentiation. However, the detailed mechanism underlying this induction remains elusive. In the current study, we report that 5-aza-CdR induces erythroid differentiation in MEL cells and BFU-E-derived erythroblasts and that this is associated with the nuclear translocation of the transcription factor c-Myc and its binding to Max to form functional complex. As markers of erythroid differentiation, the expression levels of TfR1, ferrochelatase and ALAS2 were analyzed by quantitative real-time PCR and western blotting. TfR1 and ferrochelatase expression were increased 5-fold following 5-aza-CdR treatment. To determine whether these changes in gene expression reflected differential methylation, we analyzed 1.5 kb up- and downstream from the transcription start site (TS) of these genes. Although many potential methylation sites (CpG islands) were found in both the TfR1 and ferrochelatase genes, bisulfite sequencing revealed that few sites (< 5%) were methylated in these promoters in untreated MEL cells and that there was no difference following 5-aza-CdR treatment. This observation suggests that the increased expression of TfR1 and ferrochelatase is not directly mediated by 5-aza-CdR-induced DNA demethylation. EKLF and GATA1 are key transcription regulators in erythroid differentiation, and the iron regulatory proteins (IRPs) are important for the post-transcriptional regulation of TfR1 mRNA levels. Bioinformatic analysis showed that there were many potential methylation sites in the EKLF and IRP2 promoters, however, bisulfite sequencing showed that there was no effect of 5-aza-CdR treatment at these sites in MEL cells. Since c-Myc has been reported to stimulate TfR1 expression, we then investigated the role of c-Myc in erythroid differentiation. Levels of c-Myc mRNA and protein were not increased by 5-aza-CdR, however, we did observe a dramatic increase in the nuclear fraction of c-Myc. Furthermore, co-immunopricipitation studies showed that the amount of the Myc-Max complex, which is the form in which c-Myc activates downstream gene expression, increased in the nucleus after 5-aza-CdR treatment. Interestingly, using chromatin-immunoprecipitation we also observed very strong binding of c-Myc to the CACGTG sequence upstream of the TfR1 transcription start site following 5-aza-CdR treatment. Analogous studies using primary mouse erythroblasts derived from BFU-Es gave similar results, with 5-aza-CdR treatment leading to increased TfR1 and ferrochelatase expression, c-Myc translocation to the nucleus and increased c-Myc binding activity to TfR1 promoter. Taken together, these studies point to an important role for c-Myc in the regulation of TfR1 during erythroid differentiation induced by demethylation. Precisely how demethylation is linked to alterations in c-Myc activity is under investigating. Disclosures:No relevant conflicts of interest to declare.

Molecular History of Richter Syndrome: Origin From a Common Ancestor Cell Already Present at Chronic Lymphocytic Leukemia Diagnosis

AbstractAbstract 2425Mechanisms promoting chronic lymphocytic leukemia (CLL) transformation to Richter syndrome (RS) are poorly understood and might involve antigen stimulation. We explored intraclonal diversification (ID) of immunoglobulin (IGHV) genes in RS in order to: i) follow the evolutionary history of the RS tumor clone over time; ii) understand whether antigen stimulation sustains the growth of DLBCL cells once RS is established. The study was based on 11 clonally related CLL/RS pairs, 7 clonally unrelated RS, and, for comparison, 20 de novo DLBCL. Fifty IGHV subclones were analysed per sample. Mutations observed in only one subclone were defined unconfirmed (UCM). Partially shared mutations were defined confirmed (CM). Cases were scored positive for ID only in the presence of confirmed mutations. For each sample, the normalized mutation frequency (NMF) of ongoing somatic hypermutation was calculated. Phylogenetic analyses was performed with MEGA4. Most (10/11, 90.9%) clonally related RS originated from an ancestor clone that was already present at the time of CLL diagnosis (Fig 1A, 1B, 1C). One single RS case had a transformation pattern compatible with sequential evolution from a secondary CLL subclone (Fig. 1D). All secondary CLL subclones that were documented in the CLL phase disappeared once transformation had occurred, and were substituted by the dominant DLBCL clone with its own descendants. Paired analsysis of clonally related CLL/RS samples documented that NMF significantly decreased during evolution from the CLL phase (median: 0.76 × 10−3) to the RS phase (median: 0.13 × 10−3)(p=.013). Accordingly, at transformation, the ID of IGHV genes switched off in 6/11 (54.5%) clonally related CLL/RS pairs. Clonally related RS that upon transformation maintained ID of IGHV genes were characterized by a higher prevalence of aberrant somatic hypermutation of proto-oncogenes compared to cases lacking ID (0/6 vs 3/4, 75.0%, respectively; p=.033). Also, RS cases that maintained ID of IGHV genes acquired more frequently c-MYC translocation at the time of transformation (0/6 vs 2/4, respectively; p=.133). Clonally unrelated RS are characterized by the development of a de novo DLBCL in the context of CLL. Despite morphologic and phenotypic similarities, clonally unrelated RS differed from de novo DLBCL in terms of NMF (median: 1.18 × 10−3 range: vs median: 0.08 × 10−3 range, respectively; p=.016) and prevalence of cases harboring ID (6/7, 85.7% vs 6/20, 30.0%, respectively; p=.024). The NMF was also significantly higher in clonally unrelated RS compared to clonally related cases (p=.001). These data indicate that: i) RS transformation is due to the expansion of a common ancestor clone that gains selective advantage over other subclones of the CLL phase; ii) antigen stimulation exerts different roles in clonally related RS, clonally unrelated RS and de novo DLBCL; iii) more than 50% clonally related RS switched off ID at the time of transformation, suggesting that the DLBCL clone has become independent of antigen stimulation for its sustainment; iv) clonally related RS that maintained ID of IGHV genes at transformation may take advantage of ID as a mechanism for accumulating genetic instability. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Establishment of an HS23 stromal cell-dependent myeloma cell line: fibronectin and IL-6 are critical

A multiple myeloma (MM) cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first 2 months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and, subsequently, they continued to proliferate on HS23 stromal cells. The phenotype of the established MSG1 cell line was: CD138(+), CD38(++), CD19⁻, CD56⁻, VLA-4(+), VEGFR1(+) and VEGFR2(+). Immunohistochemical staining also demonstrated expression of the IgA and λ chain in MSG1 cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities; hypertriploidy, including the deletion of chromosomes 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured only on fibronectin-coated plates with the supernatant of HS23 cells or with control medium containing IL-6. Tocilizumab, an anti-IL-6 receptor antibody, inhibited MSG1 survival under these conditions. Therefore, MSG1 may be a unique myeloma cell line that is useful for the study of cell adhesion-mediated drug resistance induced by adhesion molecules and IL-6 stimulation of myeloma cells.

The pre-B-cell receptor associated protein VpreB3 is a useful diagnostic marker for identifying c-MYC translocated lymphomas

During B-cell development, precursor B cells transiently express the pre-B-cell receptor composed of μ heavy chain complexed with VpreB and λ5 surrogate light chain polypeptides. Recent profiling studies unexpectedly revealed abundant transcripts of one member of the VpreB family, VpreB3, in a subset of mature B cells and Burkitt lymphoma. Here we used a novel antibody to investigate the normal expression pattern of VpreB3 protein in human hematopoietic and lymphoid tissues, and to determine whether VpreB3 could serve as a useful diagnostic biomarker for select B-cell lymphomas. We found that VpreB3 protein is normally expressed by precursor B cells in bone marrow and by a subset of normal germinal center B cells in secondary lymphoid organs. Among lymphoid malignancies, we found an association between VpreB3 expression and B-cell tumors with c-MYC abnormalities. VpreB3 was highly expressed in all cases of Burkitt lymphoma, whether of endemic or sporadic origin (44/44 cases, 100%), all cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5/5 cases, 100%), and the majority of diffuse large B-cell lymphomas harboring a c-MYC translocation (15/18 cases, 83%). The expression of VpreB3 in diffuse large B-cell lymphomas without a c-MYC translocation was associated with c-MYC polysomy in 25/75 cases (33%) but only rarely observed in diffuse large B-cell lymphomas lacking a c-MYC abnormality (9/98 cases, 9%). We conclude that for B-cell tumors with features suggesting a possible c-MYC translocation, such as intermediate to large cell size and high proliferation rate, the presence of VpreB3 should prompt subsequent confirmatory genetic testing, whereas the absence of VpreB3 is virtually always associated with wild-type c-MYC alleles.

Genetic and molecular mechanisms in multiple myeloma: a route to better understand disease pathogenesis and heterogeneity

Multiple myeloma (MM) is a heterogeneous plasma cell neoplasm presenting with a wide range of clinical manifestations. In spite of the availability of very performing treatment modalities, survival is highly varying, ranging from a few months to several years. Underlying genetic and microenvironmental mechanisms are thought to be responsible for clinical heterogeneity. Disease etiology is unknown but progresses in the understanding of its pathogenesis have shown that MM precursor cell transformation into a malignant one occurs in a multistep process. Possibly during class switch recombination a primary genetic event takes place. With the occurrence of additional events and the support of bone marrow microenvironmental cells, neoplastic plasma cells actively proliferate and disease behavior may change. Recurrent translocations involving the IgH locus (11q13, 4p16, 16q23, 21q12, and 6p21), deletions of chromosome 13, trisomies of chromosomes 3, 5, 9, 11, 15, 19, and 21, and dysregulated expression of cyclin D genes, are considered initiating or primary events. Alterations related to further disease transformation and adverse prognosis are deletion of 17p13, c-myc translocations, and gains of chromosome 1q21. In relation to the underlying genetic defects, disease subgroups are recognized. Accordingly treatment effectiveness may differ among groups. Intense research is ongoing in this field.

Altered Subcellular Localization of c-Myc Protein Identifies Aggressive B-cell Lymphomas Harboring a c-MYC Translocation

Nearly 100% of Burkitt lymphomas (BLs) and 5% to 8% of diffuse large B-cell lymphomas (DLBCLs) harbor a balanced translocation involving c-MYC. Although characteristic morphologic and immunophenotypic features can identify BL in most cases, tumors with atypical features are often encountered in clinical practice. Furthermore, no morphologic or immunophenotypic finding can predict an underlying c-MYC translocation in DLBCL with certainty. Here we report on a novel monoclonal antibody recognizing the c-myc protein in formalin-fixed, paraffin-embedded tissue which we used to evaluate a spectrum of aggressive B-cell lymphomas by standard immunohistochemistry. Cases consisted of 17 BLs (15 cases with confirmed c-MYC translocation), 19 DLBCLs without a c-MYC translocation, 5 DLBCLs with a c-MYC translocation, and 2 B-cell lymphomas, unclassifiable, with features intermediate between DLBCL and BL (intermediate DBLCL/BL, one case with c-MYC translocation and one case without a c-MYC translocation). The intensity and subcellular localization of tumor-specific staining for c-myc protein was determined independently by 2 pathologists and in a blinded fashion for each case. We observed c-myc expression in the tumor cells of all cases regardless of c-MYC status. Among BLs, c-myc protein primarily localized to the nucleus of tumor cells in 15 of 17 cases (88%) and equally localized to the nucleus and cytoplasm of tumor cells in 2 of 17 cases (12%). In no case did c-myc protein primarily localize to the cytoplasm. In contrast, among DLBCLs lacking a c-MYC translocation the c-myc protein primarily localized to the cytoplasm of the tumor cells in 18 of 19 cases (95%) and equally localized to the nucleus and cytoplasm in the tumor cells in 1 of 19 cases (5%). In no case did c-myc protein primarily localize to the nucleus. Among DLBCLs with a c-MYC translocation and intermediate DBLCL/BLs, the c-myc protein primarily localized to the nucleus, or equally localized to the nucleus and cytoplasm of the tumor cells in 4 of 5 cases (80%) and 2 of 2 cases (100%), respectively. Taken together, we find that a primarily nuclear or mixed nuclear and cytoplasmic staining pattern for c-myc in an aggressive B-cell lymphoma is highly predictive of a c-MYC translocation (positive-predictive value=0.92, negative-predictive value=0.95, P<0.0001). We further show that the subcellular localization of c-myc can be determined with good interobserver agreement among pathologists (kappa statistic=0.90). Thus this novel immunohistochemsitry test is a useful tool for identifying aggressive B-cell lymphomas likely to harbor a c-MYC rearrangement and thus warrant genetic testing.

T-cell leukemia 1 expression in nodal Epstein-Barr virus–negative diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma

The physiologic expression of the product of the proto-oncogene TCL1 (T-cell leukemia 1) is primarily restricted to early embryonic cells. In nonneoplastic B cells, the expression of TCL1 is determined by the differentiation step with silencing at the germinal center stage. TCL1 protein is overexpressed in a wide variety of human diseases. It has been shown that TCL1 is a powerful B-cell oncogene, which has been implicated in the pathogenesis of various types of mature B-cell lymphomas. There is no comparative information in the literature addressing the expression of TCL1 in pediatric and adult nodal diffuse large B-cell lymphoma or primary mediastinal large B-cell lymphoma. We studied 55 cases of adult and pediatric diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma to analyze the phenotypic profile of these lymphomas, including TCL1 expression, and its relationship with clinical outcome in different age groups. The cases were analyzed by immunohistochemistry for the expression of TCL1, CD10, BCL-2, BCL-6, and MUM1. We also evaluated c-MYC translocation by fluorescence in situ hybridization. TCL1 was observed in 11 cases, 5 pediatric and 6 adult cases, all but one diffuse large B-cell lymphoma. Pediatric cases showed a significant association between TCL1 expression, high proliferative index, and presence of c-MYC translocation. TCL1 positivity was predominantly found in germinal center phenotype diffuse large B-cell lymphoma. Overall survival was worse in adult TCL1-positive cases than pediatric ones. Primary mediastinal large B-cell lymphomas infrequently expressed TCL1 in both age groups.