Research in Hematopathology

Research in the Department of Hematopathology includes studies in cytogenetics, molecular pathology, leukemias, Diagnostic, immuno-phenotypic and molecular characterization of human B cell Non-Hodgkin’s Lymphomas (NHL-B), development and utilization of new immunologic and molecular tumor markers for the diagnosis of NHL-B and others.

Lynne V. Abruzzo, M.D., Ph.D.

Clinical Interests: Hematopathology and Cytogenetics

The primary focus of my laboratory is to work toward the development of a classification of lymphoid neoplasms based on patterns of gene expression at the molecular level. We hypothesize that such a classification system can be devised and that it will improve the accuracy of diagnosis and prognosis. In recent studies, we have identified conditions that give reproducible hybridizations to commercially available nylon membrane high-density cDNA microarrays. In collaboration with Dr. Kevin Coombes (Department of Biostatistics), we have developed novel statistical methods to identify and quantify components of total variability and to assess the significance of differences in relative expression levels of individual genes across multiple samples. We have evaluated multiple methods of clustering hybridization data from high-density cDNA microarrays. Furthermore, we have established that samples of malignant and normal B cells can be differentiated based on their patterns of gene expression to high-density cDNA microarrays.

One of our current studies, performed in collaboration with Drs. Michael Keating (Department of Leukemia) and Coombes, focuses on B-cell chronic lymphocytic leukemia (B-CLL), the most common leukemia in the western hemisphere. Although it is relatively morphologically homogeneous, several lines of evidence indicate that B-CLL is heterogeneous at the molecular level. We are applying the techniques that we have developed in our previous studies to subclassify B-CLL into molecular subtypes based on patterns of hybridization to high-density cDNA microarrays. Our goal is to develop a custom microarray that contains a limited number of genes and that is suitable for translation into a clinical laboratory setting to subclassify patients with B-CLL into diagnostic and prognostic groups.

Another current study, performed in collaboration with Drs. Madeleine Duvic (Department of Dermatology), Daniel Jones (Hematopathology) and Coombes, focuses on mycosis fungoides (MF), a cutaneous lymphoproliferative disorder of mature CD4-positive T cells, and Sezary syndrome (SS), its leukemic counterpart. The genetic changes underlying MF and SS are largely unknown. Our goal is to identify genes that are differentially expressed in the tumor stage of MF and SS compared with benign T cells based on their patterns of hybridization to high-density cDNA microarrays. These genes may serve as markers for diagnosis and prognosis, contribute to the understanding of the molecular pathogenesis of these disorders and identify potential therapeutic targets for these incurable diseases.

Carlos Bueso-Ramos, M.D., Ph.D.

Clinical Interests: Hematopathology - diagnostic hematopathology, immunopathology, molecular pathology. Leukemias - myelodysplasia, lymphomas

A strong interest in my lab is in factors that affect the regulation of the cell lineage determination, maturation arrest and transformation in leukemia. Current studies in my lab focus on hematopoietic cytokines and DNA binding proteins that regulate the NF-kB and p53 pathways.

Over the past 13 years, one of my primary interests has been apoptosis and proliferation in leukemias. In my work, I have shown frequent deregulation of Tp53-mdm2 and nuclear transcription factor NF-kB pathways in leukemia cells. In 1993, we performed the experiments that characterized a new gene, mouse double minute-2 (mdm2), in leukemia. Mdm2 is a key regulator of the tumor suppressor Tp53 in leukemia and other malignant neoplasms. The recognition of deregulated mdm2 expression in leukemia has led to the use of mdm2 inhibitors as leukemia therapy. Recently, I characterized mdm4, another key member of the Tp53-mdm2 pathway, in acute leukemia blast cells.

We reported high proliferation but low apoptosis in acute myelomonocytic leukemia with abnormal bone marrow eosinophils (AML-M4Eo). To assess the potential role of NF-kB genes in AML in vivo, we studied their expression patterns by oligonucleotide arrays and associated these with clinical and laboratory data to identify prognostically significant molecular subtypes. We have found dysregulated NF-kappaB activity in AML-M4Eo by molecular, immunohistochemical and flow immunophenotypic methods.

Richard J. Ford, Jr., M.D., Ph.D.

Clinical Interests: Diagnostic, immuno-phenotypic and molecular characterization of human B cell Non-Hodgkin’s Lymphomas (NHL-B), development and utilization of new immunologic and molecular tumor markers for the diagnosis of NHL-B

The studies in my laboratory continue to focus on translational research of the pathobiology of human non-Hodgkin's lymphoma (NHL) and the development of new biotherapeutic approaches for treatment of NHL. Identifying the major intracellular pathways involved in NHL cell proliferation and viability maintenance as potential targets for interdiction through new biotherapeutic approaches has recently led us to what we believe is a major new finding in lymphoma biology: the discovery that aggressive NHL-B cells have a unique method of circumventing cellular growth regulation in neoplastic B cells, through ectopic gene expression of the CD40 ligand (CD154), a major signaling mechanism for normal B cell growth and viability. This discovery has led to the realization that the CD40/CD40L/NF-kappaB signaling pathway controls NHL-B cell growth and viability through the constitutive expression of this pathway as a constant source of the centrally important transcription factor, NF-kappaB. Our studies have recently shown that NHL-B cells assemble the entire signaling pathway on an interconnected, scaffold-like signaling platform referred to as a signalosome, which is contained within a cholesterol-rich lipid membrane raft that extends into the cytoplasm from the plasma membrane. The CD40 signalosome, which contains CD40, CD154, TRAF2, IKKs and IKB-NF-kappaB, can be visualized in NHL-B cells using fluorescence confocal microscopy, where it appears as a granule-like organelle extending from the inner leaflet of the plasma membrane containing the lipid raft. The interaction of these components has been confirmed by coimmunoprecipitation studies.

Constitutive expression of NF-kappaB can be down-regulated by a monoclonal antibody to CD154, which disassembles the signalosome, blocks NHL-B cell growth and induces cell death. The signalosome can also be blocked by proteasome inhibitors that prevent the activation of NF-kappaB by its inhibitior through proteolysis in the proteasome. These findings suggest that the CD40 signalosome/proteasome axis is an excellent target for potential therapeutic agents in aggressive NHL-B. The second major translational research area relates closely to the first and involves the development of valid preclinical animal models to study the pathophysiology of NHL and to provide in vivo systems for testing the efficacy of new biotherapeutic strategies. Using fresh human biopsy specimens, we have developed xenotransplant models of the various forms of NHL in mice with severe combined immunodeficiency (SCID) disease. The NHL/SCID models closely simulate the growth patterns and dissemination (homing) pathways of the patient's lymphoma and provide excellent models to test the therapeutic effectiveness of potential biotherapeutic agents for NHL.

Daniel M. Jones, M.D., Ph.D.

Clinical Interests: Hematopathology

My laboratory is primarily focused on basic and applied clinical research in T-cell malignancies. A rotation in my laboratory would provide exposure to a variety of techniques in molecular biology and immunology. We are working on function of the TCL1 family of oncogenes that are deregulated in the mature T-cell leukemias. This group of proteins operate through modulation of serine kinases, including Akt, and through other mechanisms to drive proliferation in early stages of tumor development. This work involves proteomic techniques being done in collaboration with Ryuji Kobayashi's group in Molecular Pathology. We also working on the molecular factors underlying disease progression and the variable treatment-response of T-cell lymphoma / leukemia, using expression microarray analysis and in vivo and in vitro cell line models. We are collaborating with Nam Dang of the Lymphoma Department on these studies. We have recently focused on the role of ATM and altered regulation of the DNA repair pathways during tumor progression. Our goals are to develop better treatment-response predictors and therapeutic targets for tailored therapies in this group of tumors. The other major focus of my laboratory is on the factors mediating localization, and survival of tumor cells in cutaneous T-cell lymphoma. We are studying both CD30-positive tumor types and mycosis fungoides. This work includes studies on regulation of the chemotactic chemokines mediating tumor localization and dissemination. We are also examining T-cell receptor skewing and antigen stimulation as cofactors in early lymphomagenesis. Finally, in a collaboration with Krishna Komanduri in the Department of Blood and Marrow Transplantation, we are examining the role of lymphotropic herpesviruses as cofactors in the immune dysregulation that accompanies T-cell lymphomas.

Ming-Sheng Lee, M.D.

Clinical Interest: Hematopathology

In collaboration with our colleagues in the departments of Lymphoma and Myeloma and Radiation Oncology, members of my department and I investigated the clinical usefulness of PCR in monitoring minimal residual disease in follicular lymphoma. Upon serially analyzing more than 800 blood and bone marrow samples from 194 patients, we observed that achieving molecular complete remission (as defined by PCR negativity) during the first year of therapy is the most significant prognostic indicator in predicting longer failure-free survival (FFS) (P < 0.001). We also observed that the molecular break point of the bcl-2 gene has prognostic importance in influencing a patient's clinical outcome and FFS (P < 0.001). Moreover, we used a semiquantitative PCR assay to monitor disease progression in 55 patients. Patients with PCR values less than two arbitrary units before therapy had longer FFS than did the others (84% versus 33%; P < 0.005). Patients with PCR values of less than 0.1 arbitrary unit three to five months after treatment also had longer three-year FFS than did the others (63% versus 30%; P < 0.01). Recently, in analyzing the blood and bone marrow samples obtained from 59 previously untreated patients and correlating the results with clinical stages, we observed that patients with more advanced disease harbored a significantly higher number of cells carrying the t(14;18) translocation (P = 0.003 in bone marrow and P = 0.043 in blood).

In collaboration with our colleagues in the departments of Leukemia and Blood and Marrow Transplantation, we also developed a quantitative real-time PCR assay to monitor molecular response after treatment in patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia. A total of 396 samples obtained at different time points after treatment was studied. Of those patients, 243, 60 and 93 received IFN-based treatment, kinase inhibitor-based treatment and allogeneic bone marrow or peripheral blood stem cell transplantation, respectively. The rate and the extent of reduction in Ph-positive cells after therapy as determined by the quantities of residual Bcr/Abl fusion transcripts were significantly different in these three treatment groups. Allogeneic transplantation induced immediate and more thorough eradication of the residual Ph-positive cells: a reduction to less than 0.001% of the baseline value at two months with more than 50% of samples converted to Ph-negative sustained up to 36 months. Kinase inhibitor treatment demonstrated a rapid and substantial reduction to a mean value of 0.02% of baseline at six to 11 months of treatment. IFN-based treatment showed a slow and lesser reduction with a mean value of 0.04% of baseline, even after 36 months of follow-up care.

L. Jeffrey Medeiros, M.D.

In the research laboratory we are focusing on the biology and prognosis of anaplastic large cell lymphoma (ALCL), of which two types are currently recognized in the World Health organization: one being anaplastic lymphoma kinase (ALK)+ and the other ALK-. For in vitro studies, we are currently using a number of established ALCL cell lines, as well as cell lines derived from other non-Hodgkin lymphoma types, to investigate the biological mechanisms involved in the pathogenesis of ALCL. We are particularly working on the pathways controlling cell cycle and apoptosis. Previously we have shown that ALK+ tumors lack BCL-2 expression and exhibit a relatively higher apoptotic rate and active caspase-3 index compared with ALK- ALCL. Other BCL-2 family members are also differentially expressed between ALK+ and ALL- ALCL including BCL-XL and MCL-1 that are known targets of signal transduction pathways.

Involvement of cell-cycle regulating mechanisms in ALCL is also a focus of our studies. Recently, we have demonstrated that important check-point regulators, such as retinoblastoma protein or the cyclin-dependent kinase (CDK) inhibitor p27, appear to be deregulated in ALCL. In collaboration with Francois-Xavier Claret, Ph.D., from the Department of Molecular Therapeutics, we are working on the role of Jun activation-domain binding protein 1 (JAB1) in lymphomagenesis. Recently, a mouse JAB1 was shown to interact specifically with p27, cause translocation p27 from the nucleus to the cytoplasm and decrease p27 levels by accelerating its degradation via the ubiquitin-proteasome pathway. We are currently using gene therapy approaches (adenoviral vectors) and siRNA technology to further elucidate the pathogenetic involvement of JAB1 in lymphomas. We also have created a large clinical database of over 100 ALCL patients and have constructed high-throughput tissue microarrays of ALCL tumors. These tools allow us to rapidly assess the prognostic significance of various proteins expressed in ALCL tumors.

Francisco Vega, M.D., Ph.D.

Research Interests: Sonic hedgehog signaling pathway, lymhomas and leukemia, cancer stem cells

My research studies are centered on identifying molecular targets with promising therapeutic potential in patients with lymphomas. In particular, we are investigating the biological role of sonic hedgehog (SHH) signaling in lymphomas either as a survival or proliferative signal for the lymphoma cells or as a factor involved in self-renewal and proliferation of lymphoma-initiating cells. Currently, we are focusing on diffuse large B-cell lymphoma (DLBCL), ALK-positive anaplastic large cell lymphoma (ALCL) and chronic lymphocytic leukemia (CLL)/ small lymphocytic lymphoma (SLL).

We have shown that SHH is activated in ALK-positive ALCL and DLBCL. ALK-positive ALCL is an aggressive type of non-Hodgkin lymphoma of T-cell/null lineage characterized by chromosomal aberrations that leads to constitutive activation of ALK. The most common is t(2;5)(p23;q35), which produces a fusion between the NPM and ALK genes, leading to expression of the NPM-ALK fusion protein. We demonstrated that pharmacologic inhibition of SHH signaling as well as silencing of GLI1 gene expression by small interfering RNA decreased cell viability and clonogenicity of ALK-positive ALCL cells due to apoptosis and cell cycle arrest. We also found evidence that activation of SHH signaling in this lymphoma results from combined effects of SHH gene amplification and activation of PI3K/AKT by the kinase activity of the chimeric protein NPM-ALK, resulting in stabilization and accumulation of GLI1 protein.

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