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James Herman, M.D. || Marie Hardwick, Ph.D. ||
Robert Brodsky, M.D.
Yiping Yang, M.D., Ph.D.

Methylation PCR in Serum from Lymphoma Patients
Principal Investigator: James Herman, M.D.

Background And Preliminary Data:
Transcriptional Silencing and Methylation: Genetic alterations are a hallmark of human cancer. Changes in DNA methylation, an epigenetic modification present in mammalian cells, are also characteristic of human cancer. The CpG dinucleotide is clustered in the promoter regions of some genes. These promoter regions are called "CpG islands"; CpG islands are protected from methylation in normal cells with the exception of genes on the inactive X chromosome and imprinted genes. Methylation of promoter region CpG islands is associated with loss of expression of these genes. Promoter hypermethylation commonly silences tumor suppressor genes in human cancer, serving as an alternative mechanism for loss of tumor suppressor gene function. The p16 gene (CDKN2A) is a cyclin dependent kinase inhibitor that functions in the regulation of the phosphorylation status of the Rb protein which is frequently hypermethylated in carcinomas1-4 and lymphomas5-7. p15 (CDKN2B) also regulates the phosphorylation of Rb. Its proximity to CDKN2A leads to frequent co-deletion with p16 in the homozygous deletions observed at this locus. However, unlike CDKN2A, it is rarely inactivated by promoter region hypermethylation in most epithelial tumors with the exception of many forms of hematological malignancies including acute myelogenous leukemia and acute lymphoblastic leukemia5,8-11, Burkitt's lymphoma6,8, and in the progression of myelodysplasia to acute leukemia12,13. p15 and p16 alterations in hematological malignancy have been recently reviewed7.

Utility of determining methylation of individual genes: Genes involved in apoptotic pathways, are inactivated by promoter region methylation in cancer. We found that the death associated protein kinase (DAP kinase) is methylated in the majority of B-cell lymphomas14 and other malignancies15. Hypermethylation of the DAP kinase promoter was associated with decreased patient survival, suggesting an important role in determining the biologic aggressiveness of early-stage NSCLC16.

The DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents. Methylation of the MGMT promoter silences this gene in cancer. We recently examined whether methylation of the MGMT promoter in gliomas is related to the responsiveness of the tumor to alkylating agents17. MGMT methylation was an independent and stronger prognostic factor than age, stage, tumor grade, or performance status. The pathogenesis of diffuse large B-cell lymphoma (B-DLCL) is a heterogeneous process involving multiple molecular pathways. MGMT inactivation by transcriptional silencing due to promoter hypermethylation is a mechanism for loss of MGMT activity in B-DLCL18. 30 of 84 (36%) B-DLCL had MGMT promoter hypermethylation. The presence of MGMT methylation in lymphomas was associated with a significant increase in overall survival (hazard ratio 2.8, 95% CI, 1.2 to 7.5, p=0.01) and progression-free survival (hazard ratio 2.6, 95% CI, 1.3 to 5.8, p=0.005). MGMT promoter hypermethylation was both independent of and stronger than established prognostic factors, such as age, stage, serum LDH and performance status. The effects of MGMT promoter methylation on survival were also independent from the international prognostic indicator (IPI). (JNCI, in press, Appendix).
Promoter region methylation is a powerful molecular tool: We have recently shown that aberrant methylation of gene promoter regions is a common event in all forms of malignancy15. We analyzed a series of promoter hypermethylation changes in 12 genes (CDKN2a/p16INK4a, p15INK4b, p14ARF, p73, APC, BRCA1, hMLH1, GSTP1, MGMT, CDH1, TIMP3, and DAPK) in DNA from > 600 primary tumor samples representing 15 major tumor types. The genes play important roles in tumor suppression, cell cycle regulation, apoptosis, DNA repair, and metastasis. The hypermethylation of these genes occurs independently to the extent that a panel of three to four markers defines an abnormality in 70-90% of each cancer type. Promoter methylation represents a powerful set of markers to outline the disruption of critical pathways in tumorigenesis and for derivation of sensitive molecular detection strategies for virtually every human tumor type. In lymphoma, the most frequently hypermethylated genes are: DAP-kinase, MGMT, p16, p14 and p15.

Promoter methylation as a tumor detection marker: DNA is released and can be detected in the serum of patients with cancer. The level of this DNA has been somewhat correlated to tumor burden. Tumor specific alterations in DNA can be detected and the specificity of these changes allows the separation of DNA from tumor and normal tissue. We have developed and sensitive and specific method of detection for methylation changes19. Promoter region methylation has been detected in the serum of patients with lung, head and neck, breast, and colon cancer20-22. The presence or level of this tumor specific change may correlate with disease status and a reduction in tumor DNA may reflect tumor response. In a recent study of esophageal cancer, hypermethylated APC DNA was observed in the plasma of 25% adenocarcinoma patients and high plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival23. Similar results have been reported for non-small cell lung cancer24.

Multi-gene analysis: To analyze a number of genes and to work with samples with limited amounts of DNA from formalin-fixed paraffin-embedded DNA we have developed a multiplex nested MSP approach. The sensitivity of this approach has been enhanced approximately 50 fold. The specificity of MSP is maintained during this modification. We have already combined the external primers for 17 candidate genes and tested the specificity of MSP for these genes. All genes retained the specificity of the original MSP procedure (example Figure 1).

Figure 1. Methylation specific PCR for the p15 and p16 genes following muliplex nested approach. Normal lymphocyte DNA (Nl) shows no methylation of these genes, as shown by the lack of PCR product in lanes labeled M, while visible signal is present in the U lane. Control DNA demonstrates amplification in the M lane. Methylated control DNA was mixed with normal DNA at the ratio shown above, and M signal is positive for all dilutions, even to 1:100000 (that is 10 pg methylated DNA mixed in 1 microgram of normal lymphocyte DNA), demonstrating the sensitivity and specificity of this approach.
Overall Approach/Study Design: This will be prospective cohort study. Detection of aberrant methylation of promoter regions by methylation-specific PCR will be used. A multiplex-nested MSP analysis as described above will be employed. Tumor specimens will be used to determine a methylation profile of each tumor. Serum will be collected during routine blood sampling during clinic visits. CT scans will be used to determine response to therapy or relapse. DNA extracted from serum or plasma assayed to determine the presence or absence of tumor specific DNA alterations in the patient’s blood. Molecular results will be correlated with disease state

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Bcl-2 Point Mutations            

Principal Investigator: Marie Hardwick, Ph.D.

Introduction and Rationale
The bcl-2 oncogene was first identified at t(14;18) translocation breakpoints that occur in the majority of individuals with follicular B cell lymphomas (22,23). This translocation event results in the overexpression of bcl-2 that allows B cells to survive when they would normally die by apoptosis (12,14). Overexpression of the Bcl-2 protein protects a wide variety of cell types from many death-inducing stimuli including growth factor withdrawal/axotomy, treatment with calcium ionophores, glucose withdrawal, membrane peroxidation, glucocorticoid treatment, chemotherapeutic agents, and virus infection, implying that BCL-2 functions at the point of convergence of many different signals (1). A role for Bcl-2 as a true physiologic inhibitor of cell death in animals is supported by studies with knockout mice. In Bcl-2-deficient mice, the immune system starts to develop normally, but massive apoptosis in the spleen and thymus occurs subsequently (10,16,17,24). More recently, altered expression of the BCL-2 protein has been described in a wide range of human cancers, without the occurrence of chromosome translocations. Additionally, a body of evidence indicates that elevated BCL-2 expression causes resistance to chemotherapeutic treatments while suppressed BCL-2 expression correlates with apoptosis in response to anticancer agents (18). Therefore, anticancer strategies that target BCL-2 by anti-sense mechanisms or peptides/drugs that bind in the cleft of BCL-2 are being explored. How does Bcl-2 block apoptosis? The biochemical mechanism by which BCL-2 inhibits apoptosis is still not fully understood but BCL-2 is generally believed to work at the mitochondrial membrane where it prevents mitochondrial damage that otherwise leads to leakage of prodeath mitochondrial factors into the cytosol and eventual disruption of mitochondrial membrane potential (3).

We have shown that endogenous BCL-2 is cleaved in the loop domain near its N-terminus by caspase-3 during programmed cell death in a variety of cell types (2). The resulting C-terminal cleavage product localizes to mitochondria, induces the release of cytochrome c and is potently pro-apoptotic (2,11). It has been suggested that cleavage of BCL-2 is important in chemotherapy-induced death of leukemia/lymphoma cells (7,8). This phenomenon where prodeath activity is induced or enhanced following proteolysis is now known to occur for many BCL-2 family members including BCL-xL, BID, BAX and BAD (4,9,13,20). Sequencing of six cell lines and tumors derived from non-Hodgkin’s lymphomas revealed that one of these contained a point mutation in the caspase cleavage site at Asp34 as well as other mutations (19,21). The aspartate residue at the P1 position (amino acid 34) is absolutely required for caspase cleavage. Therefore, the BCL-2 protein in this tumor cell would not be susceptible to caspase cleavage. Mutation of the caspase cleavage site in BCL-2 is known to significantly enhance the antiapoptotic function of BCL-2 rendering these cells less susceptible to a variety of death stimuli including ionizing radiation (2,4). Therefore, we propose to examine the sequence of BCL-2 found in a bank of lymphoma samples to explore the possibility that modified susceptibility of BCL-2 to proteolysis contributes to lymphoma. In particular we will focus on relapsed patients are these are deemed most likely to contain such mutations.

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Augmentation of antibody-mediated complement dependent cytotoxicity in lymphoma.

Principal Investiagtor: Robert Brodsky, M.D.

Monoclonal antibodies are effective in the treatment of a wide variety of lymphoproliferative disorders and autoimmune diseases. It is now recognized that monoclonal antibodies kill cells through a combination of complement-mediated cytotoxicity, antibody-dependent cellular toxicity, and apoptosis.1-3 Recent data demonstrate that a major cause of resistance to monoclonal antibody therapy is an upregulation of the complement-regulatory proteins, CD59 and CD55.2;4

The human disease paroxysmal nocturnal hemoglobinuria (PNH) is clonal hematopoietic stem cell disorder that leads to a marked deficiency or absence of the glycosylphosphatidylinositol (GPI) anchored complement regulatory proteins, CD55 and CD59.5 Our preliminary data demonstrates that EBV-transformed lymphocytes from PNH patients are markedly more sensitive to monoclonal antibody therapy and that selective biochemical removal of GPI-anchored proteins greatly increases complement-mediated cytotoxicity in non-PNH lymphoma cell lines. Thus, we propose to investigate molecular, biochemical, and pharmacologic strategies to interrupt GPI-anchored proteins in order to augment antibody-mediated complement dependent cytotoxicity in lymphomas.

Specific Aims:

1. Disruption of GPI-anchor protein expression will greatly increase the sensitivity of lymphomas to monoclonal antibody therapy.
2. Establish preclinical models to test the efficacy of GPI-achor ablation in treating lymphomas.
Hypothesis 2.1: Lymphomas devoid of GPI-anchors will be more vulnerable to monclonal antibody therapy than GPI-anchor replete lymphomas.
Hypothesis 2.2: PIPLC can safely and transiently remove GPI-anchored proteins in bone marrow specimens resulting in more effective purging with monoclonal antibodies.

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Antigen-defined Immunotherapy for Lymphoma: The Use of Lentivirus transduced Dendritic Cells Matured ex vivo or in vivo
Principle Investigator: Yiping Yang, M.D., Ph.D., Co-Investigator: Drew Pardoll, M.D., Ph.D.

Background, Rationale And Specific Aims
The major barrier to cancer immunotherapy has been immune tolerance to tumor antigens (1). A potent vaccine capable of inducing effective anti-tumor immunity is desired to reverse immune tolerance to these tumor antigens. Dendritic cell (DC)-based vaccines have emerged as a promising approach for eliciting effective anti-tumor immune responses. To be effective, these peptide, protein or RNA loaded DC, or virally transduced DC have to be administered before or a few days after tumor challenge (2-5). This appears to be due, in part, to inefficient loading of tumor antigens (via retrovirus or RNA), or lack of CD4 T helper function in the case of CD8 peptide loading or lack of sustained antigen presentation by DC (via protein loading). These factors limit the effective and sustained stimulation of antigen specific T cells by DC. In an attempt to improve DC-based vaccine strategies, we have developed a different approach to allow more efficient tumor antigen presentation by lentivirus-transduced DC matured ex vivo or in vivo in a murine model of A20 B cell lymphoma using HA as a model tumor antigen. HA behaves like a natural tumor antigen in a number of tumor models such as the A20 lymphoma, in that moderate levels of HA expression do not alter the biology, immunogenicity, or in vivo growth characteristics of the tumor (6-9). In particular, the groups of Dr. Pardoll and Dr. Levitsky at Johns Hopkins have extensively utilized the HA antigen and the pair of MHC class I (Kd) and II (I-Ed) restricted, HA specific TCR transgenic mice to address fundamental mechanisms of immunoregulation.
The goal of this application is to explore the utility of lentivirus-HA transduced DC matured ex vivo or in vivo in eliciting effective sustained antigen-specific anti-tumor immunity. In particular, we will evaluate whether vaccination with mature DC transduced with self-inactivating (SIN) lentiviral vectors (ref. 10) expressing HA generates effective antigen-specific T responses to “break tolerance” in an HA tolerant transgenic mouse model. In parallel, we will explore a novel approach to allow efficient tumor antigen presentation by DC derived from hematopoietic stem cells (HSC) transduced with lentivirus HA and selectively activated in vivo with a drug inducible DC activator. Success in enhancement of antigen specific T cell activation against tumor cells by these novel approaches will lead to clinical trials in treating antigen-defined tumors such as EBV (+) Hodgkin’s lymphoma. We will focus on the following specific aims:
1) To evaluate the ability of lentivirus-HA transduced DC vaccine to “break tolerance” in HA tolerant transgenic mouse models.
2) To investigate whether lentivirus-HA transduced HSC-derived DC selectively activated in vivo with a drug inducible DC activator, induce antigen specific T cell activation in mature T cells.
3) To assess anti-tumor efficacy of these different approaches in murine prevention and treatment models using the well characterized HA-expressing A20 lymphoma

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