Career Development Awards
Genetic Abnormalities and Ectopic BCL-10 Expression in Extranodal Marginal Zone B-Cell Lymphomas of Mucosa-Associated Lymphoid Tissue (Malt Lymphomas): Analysis By Fluorescence in Situ Hybridization, Molecular Genetics, and Immunohistochemistry
Investigator: Ellen Remstein, M.D., Mayo Clinic
Dr. Remstein focused her work on genetic abnormalities in low-grade lymphomas of mucosa-associated lymphoid tissue (MALT lymphomas). Her initial studies, which examined primary pulmonary MALT lymphomas, demonstrated that cytogenetic abnormalities were common (75 percent) and heterogeneous.
In a separate publication she compared detection of the 14;18 translocation in follicular non-hodgkins lymphoma (NHL) using the routine polymerase chain reaction (PCR) with FISH in paraffin-embedded tissue samples. FISH was found to be superior to PCR for detecting the 14;18 translocation. Dr. Remstein pooled her cases of MALT lymphomas with other investigators for a joint publication in the Journal of Pathology in 2005.
In collaboration with Grzegorz Nowakowski, M.D., Dr. Remstein studied the clinical utility of FISH for IgH translocations in 1,032 patients with chronic lymphocytic lymphoma (CLL). They found 76 (7 percent) patients had an IgH translocation. She had another success with Ahmet Dogan, M.D., Ph.D., working to identify unknown partner genes by long distance inverse PCR in MALT lymphoma cases in which the IgH translocations had previously been identified by FISH but in which the partner gene was unknown. This work has continued through support via a UI/MC Developmental Research Program Award.
Another outcome of her research under this Career Development Award has been an improvement in cytogenetic techniques. Dr. Remstein's work has led to modifications of standardized techniques that enable karyotypes to be obtained from a variety of different B-cell lymphomas. She has increased the ability to perform in particular, cytogenetic analyses by preicubating lymphoma specimens with CpG.
The idea of using CpG ODN for this purpose resulted from discussions related to the UI/MC SPORE, and represents an important technical advance that will impact on lymphoma research and clinical practice. For example, use of this new method has led to the discovery of new balanced translocations in several B-cell lymphomas, including MALT lymphomas and splenic marginal zone lymphomas.
Taken together, Dr. Remstein's research findings have been pivotal in identifying new chromosomal abnormalities in MALT lymphomas. This has improved researchers' understanding of the genetic mechanisms of these tumors and improved diagnostic classification. This FISH strategy has now moved into testing in the North Central Cancer Treatment Group (NCCTG) as a component of the translational research in the ongoing clinical trial N0489 (RE-CHOP for large cell lymphoma). Lastly, she has discovered a new chromosomal partner that has led to a new developmental project in the SPORE renewal.
Dendritic Cell Generation and Immunobiology
Investigator: Thomas Warren, M.D., University of Iowa
Preclinical Development of Adaphostin for Treatment of Patients with Chronic Lymphocytic Leukemia and Non-Hodgkin's Lymphoma
Investigator: Tait Shanafelt, M.D., Mayo Clinic
The focus of Dr. Shanafelt's research is to develop novel agents for small lymphocytic lymphoma (SLL)/CLL, and NHL in general.
Tyrphostins are a class of small molecules that were designed to act as tyrosine kinase inhibitors but have been shown to induce apoptosis through elevation of reactive oxygen species (ROS). Dr. Shanafelt has demonstrated that one of the tyrphostins, adaphostin, could induce apoptosis of SLL/CLL cells in vitro. He is therefore continuing with the pre-clinical development of ROS-generating agents for treatment of low grade B-cell NHL with the goal of moving this class of agents into the clinic. Because it was not known whether adaphostin would ever enter human testing, Dr. Shanafelt re-focused his work to study other ROS-generating agents such as the green tea extract epigallocatechin-3-gallate (EGCG) on primary lymphoma cells and lymphoma cell lines. His work demonstrated the activity of this agent against lymphoma cell lines and primary SLL/CLL B-cells in vitro.
He has now completed additional testing of EGCG on primary human lymphoma cells and lymphoma cell lines demonstrating this agent is active against a wide range of non-Hodgkin lymphoma cells (manuscript under review). This work has led to a Phase I clinical trial entitled "A Phase I/II Study of Daily Oral Polyphenon E in Asymptomatic, Rai Stage 0-II Patients with Chronic Lymphocytic Leukemia." Depending on the results of this trial, additional trials of this agent in other lymphoma subtypes may be warranted. Dr. Shanafelt is also studying whether combinations of EGCG with common chemotherapy agents and monoclonal antibodies can be synergistic in their anti-tumor activity.
- Suppressors of cytokine signaling proteins in human hematopoietic cancers
Investigator: John Colgan, Ph.D.
Investigating the expression and potential role of the cell cycle inhibitor, cyclin G2, in non-Hodgkin B-cell lymphomas
Investigator: Mary Horne, Ph.D.
Increasing the efficacy of alemtuzumab and rituximab in CLL
Investigator: Clive Zent, M.D.
In this project, Dr. Zent and his colleagues sought to study the mechanism of action of monoclonal antibodies (MoAb) in readily-accessible primary tumor cells of chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL).
Using the knowledge that rituximab and alemtuzumab (Campath-1H) result in high response rates even in high risk CLL—but no cures, they investigated the mechanisms of these two moAbs to see if their combination might be more effective. They found that the combination of these two antibodies was highly active in patients with high risk, early stage CLL with responses in 90 percent of patients (n=30). Correlative laboratory studies showed that alemtuzumab consistently kills approximately 70 percent of CLL cells by complement dependent cytotoxicity (CDC). In contrast, rituximab does not cause CDC of CLL cells in these assays.
Based on the hypothesis that the effectiveness of both alemtuzumab and rituximab would be increased by the use of drugs that are known to enhance antibody dependent cellular cytotoxicity (ADCC) such as β-glucan, GM-CSF and CpG 7909, Dr. Zent proposed to use leukemic CLL B-cells from early stage patients in order to:
- Measure alemtuzumab- and rituximab-induced binding of iC3b to CLL cells and establish dose response curves for each drug alone and in combination
- Determine if autologous neutrophils are cytotoxic to iC3b-coated CLL cells in the presence and absence of β-glucan and establish dose response curves for β-glucan
- Determine if GM-CSF can increase ADCC and CR3-dependent cellular cytotoxicity against CLL cells by:
- Testing the cytotoxicity of autologous granulocytes and monocytes pre-treated with GM-CSF
- Testing if GM-CSF increases the metabolism of the oral preparation of β-glucan to the active metabolite
- Determine the effect of CpG 7909 on the cytotoxicity/apoptosis potential of the combination of MoAb, complement, β-glucan, and GM-CSF for CLL B cells
He found that alemtuzumab does kill a large percentage of CLL cells by complement dependent cytotoxicity but that this did not predict response to treatment. This finding suggests an important role for antibody dependent cytotoxicity. This mechanism continues to be investigated in ongoing studies. In addition, cells surviving alemtuzumab-mediated complement dependent cytotoxicity were found to have a high level of expression of iC3b which is a target for cellular cytotoxicity that can be increased by yeast-derived β-glucan. This continues to be tested in ongoing studies. The results of these studies have led to the development of a trial using pentostatin, alemtuzumab, and low dose rituximab for treatment of relapsed/refractory CLL.