Overall Abstract

The University of Iowa/Mayo Clinic Lymphoma SPORE (UI/MC SPORE) is a highly successful translational research program that takes advantage of the combined strengths of the translational lymphoma programs of the Holden Comprehensive Cancer Center at the University of Iowa and the Mayo Clinic Comprehensive Cancer Center. Both centers have extensive experience in lymphoma research extending from basic investigation through performance of innovative clinical trials. These two institutions have a long history of collaborating on studies focused on the epidemiology of lymphoma. Scientific accomplishments include translational studies exploring the potential of a novel therapeutic agent, immunostimulatory CpG ODN, as a potential treatment for B cell malignancies, development of novel approaches to imaging that could impact on our understanding of lymphoma biology, and investigation into biomarkers that could have a significant impact on management of lymphoma.

The UI/MC SPORE consists of 4 research projects, 4 core resources, and the Career Development and Developmental Research Programs. Specific projects are as follows:

1. A novel approach to the immunotherapy of B cell malignancies
2. Signal transduction inhibitor therapy for lymphoma
3. Biology and Epidemiology of APRIL and BLyS in B-cell NHL
4. Regulatory T-cells in the tumor microenvironment of B-cell non-hodgkin lymphoma

Core resources include Administration, Biostatistics and Bioinformatics, Biospecimens, and Clinical Research that supports both clinical trials and the Molecular Epidemiology Resource of the UI/MC SPORE. All units within the UI/MC SPORE work to draw on the resources of both institutions to expedite the translation of discoveries into new and better approaches to the prevention and treatment of lymphoma.

Project 1: A Novel Approach to the Immunotherapy of B Cell Malignancy
Principal Investigator: George J. Weiner, M.D. (Iowa)
Co-Principal Investigator:  Clive Zent, M.D.  (Mayo)
Co-Investigators: Zuhair Ballas, M.D. and Brian Link, M.D. (Iowa)

Studies have demonstrated that immunostimulatory CpG ODN and IL-21 are synergistic in their ability to induce apoptosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) cells, and that this apoptosis is independent of benign cells. One of the mechanisms responsible for this apoptosis is production of Granzyme B by the malignant B cells – a surprising finding given that B cells have not previously been shown to produce Granzyme B. It was also found that other B cells can be induced to produce Granzyme B. Further studies demonstrated Granzyme B production by B cells can also be induced by IL-21 plus anti-B cell receptor antibody (Anti-BCR). The identification of a potentially powerful new mechanism of anti-tumor activity opens up a number of new avenues for investigation.  This project will continue to explore the mechanisms responsible for induction of Granzyme B production by the B cells, how cells treated in such a manner mediate anti-tumor activity, and how such activity can be utilized therapeutically.  These studies, which will make extensive use of all of the UI/MC SPORE shared resources, will provide valuable information on how the unexpected immunologic finding that B cells can produce functional Granzyme B, can be applied to the treatment of Lymphoma and other B cell malignancies.

The two specific aims of this project are:

Aim 1: Assess the effects of IL-21, CpG ODN, and other B cell activators, on malignant B cells in vitro.

• Define the molecular changes induced by IL-21, CpG ODN and anti-BCR, alone and in combination, and correlate these changes with the effect of therapy on malignant B cell viability and phenotype.
• Delineate the relative importance of autolysis, undirected cytotoxicity and antibody directed cellular, cytotoxicity (ADCC) in the apoptosis of malignant B cells treated with IL-21 plus CpG ODN or IL-21 plus anti-BCR.
• Assess how malignant B cells respond to other combinations of B cell activating agents.

Aim 2:  Perform early phase clinical trials of biological therapy in subjects with CLL and assess the impact of treatment on the biology of CLL cells.

• Assess whether clinical therapy of CLL subjects with CpG ODN induces changes in CLL cells similar to those observed in vitro, and how dose and route of CpG ODN impact on this response.
• Evaluate how clinical therapy with CpG ODN impacts on the response of CLL cells to IL-21, and other biological agents in vitro.
• Conduct additional clinical trials of combinations of biologically active agents in CLL or other B cell malignancies based on the initial preclinical and clinical results.

Project 2: Signal Transduction Inhibitor Therapy for Lymphoma

Principal Investigator: Thomas Witzig, M.D. (Mayo)
Co-Principal Investigator: Scott Kaufmann, M.D., Ph.D. (Mayo)
Co-Investigators: Raymond Hohl, M.D., Ph.D. (Iowa); Patrick Johnston, M.D., Ph.D. and Shaji Kumar, M.D., (Mayo)

Lymphomas are the fifth most common neoplasms in the United States, with nearly 80,000 new cases of non-Hodgkin lymphoma (NHL) and Hodgkin disease (HD) each year.  Treatment advances over the last several decades have improved the survival of patients with these common malignancies.  However, nearly 40% of patients with large cell NHL, 80% with indolent NHL, and 20% with HD are not cured and die of their disease.  New agents with unique mechanisms of action based on knowledge of signal transduction pathways in lymphoma cells are needed to advance lymphoma treatment.  This project will focus on the phosphatidylinositol-3 kinase (P13K) and Raf kinase pathways in lymphoma cells.  The overall hypothesis is that a combination of chemotherapy agents with one or more of the signal transduction inhibitors (STIs) will improve the response rate and survival of patients with NHL/HD.  To test this hypothesis, we will include clinical trails that assess rational combinations of STIs with each other and with conventional chemotherapy agents, investigational biomarkers in lymphoma cells from patients participating in these trials, and in vitro studies of new agents and combinations in primary tumor cells that will lead to the next generation of clinical trials including study of combinations with substantial clinical activity in the cooperative groups.

This work is organized in 3 specific aims:

Aim 1:  To investigate the safety and efficacy of PI3K/Akt/mTOR pathway inhibitors in combination with Raf-kinase inhibitors and conventional chemotherapy agents. The SPORE will conduct two separate trials: 1) a Phase II trial of tipifarnib plus etoposide for relapsed aggressive NHL at Iowa/Mayo, and 2) a phase I/II trial of sorafenib plus everolimus for B-cell malignancies that will be performed as an InterSPORE trial between this Lymphoma SPORE and the Dana Farber Myeloma SPORE.

Aim 2:  To assess the action of combinations of STIs on the targeted pathways and identify potential markers of anti-tumor efficacy using malignant B-cells from patients entered on the trials in Aim 1. Because tumor response will be heterogeneous, we will utilize tissue samples from patients entering the SPORE trials to assess the impact of treatment on levels of expression of key members of the PI3K and Raf kinase pathways, tumor cell proliferation/apoptosis, and levels of tumor angiogenesis.

Aim 3: To investigate novel combinations containing agents targeting the PI3K/Akt/mTOR pathway and other STIs or conventional agents in malignant B-cells in vitro to provide the rationale for the next generation of clinical trials. Malignant B-cell lines will be used to test combinations of STIs with each other and with conventional chemotherapy agents known to have activity in the B-cell malignancies. Promising combinations will be tested on fresh primary malignant B-cells from patients accrued to the Biospecimens Core. Our initial studies will focus on drugs that target PI3K/Akt/mTOR pathway components or those of pathways known to connect with the PI3K/Akt/mTOR pathway. Studies will also include compounds being studied through the current SPORE Developmental and Career Development Research programs.

Co-Principal Investigators: James R. Cerhan, M.D., Ph.D. and Anne Novak, Ph.D. (Mayo)
Co-Investigators:  Gail Bishop, Ph.D., and Brian Link, M.D., (Iowa):  Stephen Ansell, M.D., Ph.D., and Richard Bram, M.D., Ph.D., (Mayo)

There is accumulating evidence that implicate the TNF superfamily members BLyS and APRIL, as well as their receptors, as critical factors for the growth and survival of both normal and malignant B cells.  BLyS, which is better characterized than APRIL, has been found to be elevated in a number of immune disease models and there is increasing evidence that it may correlate with pathogenesis of various B cell related disorders, including B cell malignancies.  BLyS and APRIL are expressed in B-cell non-Hodgkin lymphoma (NHL) and the expression of BLyS is associated with an aggressive disease phenotype.  While it is clear that BLyS expression is required for normal B cell development and homeostasis, the exact source of BLyS in the normal and malignant scenario remains to be fully elucidated.  Because serum BLyS levels are elevated in a number of B cell malignancies known to have a familial incidence, it is possible that dysregulation of BLyS occurs at the genetic level.  The environmental, as well as genetic, requirements that mediate BLyS expression remain to be defined, and the promoter for the BLyS gene is poorly characterized.  In preliminary work generated from our UI/MC Lymphoma SPORE Developmental Projects, we have found that a polymorphism in the BLyS promoter region correlates with increased serum BLyS levels in patients with B-cell malignancies, particularly those with a family history of B-cell related cancers.   We now propose to follow-up these findings through a new, integrated basic and population science project that utilizes the specimen and epidemiology resources developed through the UI/MC Lymphoma SPORE Biospecimens Core and the Molecular Epidemiology Resource during the first project period.  We will determine if genetic variability in BLyS, the BLyS receptors TACI, BCMA, and BAFF-R, as well as the BLyS related TNF molecule APRIL, are associated with the development of NHL and the clinical outcome of patients.  In addition to our genetic studies we also propose to determine the role of APRIL on the biology of NHL B cells.  We hypothesize that APRIL is involved in the growth and survival of malignant B cells and may contribute to the pathogenesis of NHL.  Identification of patients who have or are predisposed to elevated BLyS and APRIL levels, or those who have genetic alterations in BLyS, APRIL or their receptors, will provide us with an opportunity to better understand the significance of these molecules in B cell malignancies and ultimately to translate these findings to improved clinical management and perhaps novel therapeutic approaches.  To address these questions we propose the following aims:

Aim 1. To evaluate the association of inherited variability in genes encoding BLyS, APRIL, and their key receptors, with risk of NHL.

Aim 2. To evaluate the association of inherited variability in genes encoding BLyS, APRIL, and their key receptors, with NHL survival.

Aim 3. To functionally characterize the BLyS promoter polymorphisms and identify inflammatory mediators involved in BLyS expression.

Aim 4. To determine the significance of APRIL and its receptors in the survival and proliferation of NHL B cells.

Project 4: Regulatory T-Cells in the Tumor Microenvironment of B-Cell Non-Hodgkin Lymphoma

Principal Investigator: Stephen Ansell, M.D., Ph.D. (Mayo)
Co-Investigators:  Brian Link, M.D., (Iowa); Richard Bram, M.D., Ph.D., and Keith Knutson, Ph.D. (Mayo)

B-cell non-Hodgkin lymphomas (NHL) are common lymphoid cancers in which malignant cells arrested at various stages of differentiation proliferate within lymph nodes and occasionally other tissues. However, cells other than tumor cells are commonly present in the tumor microenvironment. These cells include T lymphocytes that seem to be more than simple residual elements from the normal lymph node structure. It is commonly believed that these infiltrating immune cells are targeting the cancer cells, yet they appear unable to eradicate the malignant cells. Despite extensive studies regarding anti-tumor immunity, the significance of infiltrating T cells in B-cell NHL remains poorly understood.

Recent studies in other cancers have suggested that regulatory T (Treg) cells are involved in the control of anti-tumor immunity by inducing tolerance to the tumor. It has been shown that Treg cells influence tumor immune responses by suppressing tumor-specific immune cells. However, there are little data regarding the effect of Treg cells on tumor-specific T cell immunity in B-cell NHL and subsequently on the malignant B-cell growth. Studies in B-cell lymphoma have described a T-cell or immune signature in the tumor that correlates with patient outcome. The immune infiltrate is usually comprised of CD4+ and CD8+ T-cells as well as monocytes.

Our hypothesis is that intratumoral Tregcells in patients with B-cell NHL significantly upregulate their ability to suppress tumor specific T-cell responses as they enter sites infiltrated by malignant B-cells. We further postulate that malignant B-cells play an active role in this process by direct activation of Treg cells thereby facilitating immune tolerance to their presence.

To determine whether localization of Treg cells influences their suppressive capacity and to define the role of malignant B-cells in the migration and activation of Treg cells in patients with B-cell lymphoma, we propose the following aims –

Aim 1:To show that Treg cells are pathologically recruited to areas of B-cell NHL and gain suppressive function when present in the malignant lymph nodes.

Aim 2: To determine whether malignant B-cells directly interact with Treg cells to facilitate immune tolerance.

Aim 3: To test whether therapeutic inhibition of Treg cell recruitment and function results in significant clinical benefit for patients with B-cell NHL

Core 1:  Administration

Director:  George J. Weiner, M.D. (Iowa)
Co-Director: Thomas Witzig, M.D. (Mayo)

The overall goal of the University of Iowa/Mayo Clinic Lymphoma SPORE (UI/MC SPORE) Administration Core is to stimulate research in lymphoma and to expedite the translation of discoveries into new and better methods of prevention, detection, and treatment of lymphoma.  The Administration Core serves as the organizational hub of the UI/MC SPORE.  It provides an organizational structure designed to efficiently coordinate the activities of the research projects, scientific cores, and developmental programs (Developmental Research Program, Career Development Program) of the UI/MC SPORE.  It also serves to enhance communication between investigators at Iowa and Mayo.  The Administration Core is responsible for coordinating the function of the SPORE committees including the Executive Committee and the External Advisory Committee.  Dr. George Weiner will serve as Director of the Administration Core, and Dr. Thomas Witzig as Co-Director.  Drs. Weiner and Witzig will continue to work collaboratively to provide expertise in laboratory and clinical lymphoma research.  They have extensive administrative experience and are well qualified to provide leadership and direction for the UI/MC SPORE.  The Administration Core will support SPORE activities by: (1) providing leadership, organizational support, and financial management for UI/MC SPORE investigators; (2) coordinating monthly videoconferences and quarterly meetings of UI/MC SPORE investigators, and the ongoing scientific review of SPORE research projects and cores, and the visits of the external advisors; (3) providing the infrastructure for the selection and support of the best and most promising projects as outlined in SPORE guidelines; (4) providing for information transfer to the scientific community via professional and public means; (5) providing the structure for the establishment and nurture of collaborations to facilitate and expand lymphoma research; (6) assuring input from patient advocates and (7) fostering trainee development.

Director: Ahmet Dogan, M.D., Ph.D. (Mayo)
Co-Director: Sergei Syrbu, M.D., Ph.D. (Iowa)

The UI/MC SPORE Biospecimens Core provides a coordinated, centralized, and dedicated Core for the procurement, processing and annotation of biospecimens from lymphoma patients and patients with small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL).  The goal of the Biospecimens Core is to procure a variety of biologic specimens on all patients involved in UI/MC SPORE protocols and all newly diagnosed lymphoma patients seen at the University of Iowa and the Mayo Clinic Rochester.  The specific aims of the Biospecimens Core are:  1) to collect, process, distribute, and bank cells from fresh tumor, frozen tumor tissue, paraffin-embedded tumor tissue, serum/plasma, and genomic DNA from lymphoma and SLL/CLL patients; 2) to track all biospecimens in the Biospecimens Tracking Database; and 3) to serve as a resource of expertise, collaborative support (including other NIH grants), and service for projects involving pathology review/classification, immunohistochemistry, in situ hybridization, fluorescent in situ hybridization, laser capture microdissection, tissue arrays, and digital image analysis.  All specimens are collected and processed under tight quality control, and distributed to UI/MC SPORE researchers or banked for future SPORE research projects.  These activities are tracked using a sophisticated database that merges the activities at Iowa and Mayo, and allow integration with clinical and other data collected in research projects.  , Over 1300 newly diagnosed lymphoma patients and over 350 previously diagnosed lymphoma patients for use of pathology tissues have been consented.  In most of these patients we have banked serum and DNA, and on over 500 patients we have processed and banked cells from fresh tissue available after clinical work-up.  The Core has supported multiple full and developmental projects, as well as Career Development awardees.  The Core has also partnered with other projects that have obtained extramural funding to support epidemiologic and family studies, and has provided key infrastructure for the Molecular Epidemiology Resource.  We will continue to accrue new patient samples to the bank and work with investigators to utilize this increasingly valuable resource to support translational research projects in lymphoma.

Director: Susan M. Geyer, M.D., (Mayo)
Co-Director: Terry Braun, Ph.D., and Brian Smith, Ph.D. (Iowa)

The Biostatistics and Bioinformatics Core (BABC) provides statistical collaboration and data management support for each of the SPORE projects, the developmental projects, and the other Cores. In addition, the BABC also provides collaboration and support in bioinformatics and computational biology research for SPORE projects as appropriate. The BABC established the infrastructure to link the Lymphoma clinical and research databases between UI and MC. This system is fully functional and allows web-based registration and data entry from both sites into a common database.  This core provides data management for clinical trials, monitors adverse events in collaboration with the Clinical Research Core, and prepares data summaries for manuscript preparation. The BABC has been very active in preparing the statistical plan for each of the four Projects.  Each of the projects presented in this application reflects input from members of the BABC on study design, analysis plan, and bioinformatics needs.  These projects span a wide range of approaches and analyses required. The BABC builds upon the innovative and time-tested procedures and systems developed by Mayo Clinic, one of the largest statistical groups in the country whose members have collaborated on more than 8,000 clinical and basic science research studies since 1966, as well as the Coordinated Laboratory for Computational Genomics at the University of Iowa founded in 1996. The BABC will provide statistical and bioinformatics support across different fields, including epidemiological studies, basic sciences including translational and immunologic correlative studies, gene microarray and imaging, clinical trials, and gene and mutation discovery, expression analysis and genomics, and information management.  The comprehensive nature of the BABC, which will have activities at both the MC and UI, assures each SPORE investigator access to statistical and bioinformatics expertise that includes collaborative development of study designs and analysis plans, state of the art data analysis and interpretation, data management resources, and abstract and manuscript preparation. The BABC also provides a mechanism for the management and integration of both existing and newly collected data through consistent and compatible data handling. Areas of support include database development, data form development and processing, data collection and entry, data archiving, quality control, and management of information relating to gene mutation identification and genotyping data for disease linkage experiments. This Core complements and assists the efforts of the Clinical Research and Biospecimens Cores by providing superior data management and experience with tissue registries. The strengths of the BABC are our collaboration with each of the projects and cores, the ability to utilize the established centralized research database as well as the operational and statistical infrastructure already in place in the UI/MC Lymphoma SPORE, and the breadth of expertise provided by BABC personnel.

Core 4: Clinical Research

Co-Director:  Brian Link, M.D. (Iowa) and Thomas Habermann, M.D. (Mayo)
Co-Investigator:  Patrick Johnston, M.D., Ph.D. (Mayo)

The UI/MC SPORE Clinical Research Core (CRC) has as its primary goal to be the direct translational link between research projects and clinical research emanating from these projects. The CRC coordinates the development of clinical trials, assists in patient accrual, manages and reports adverse events to appropriate agencies, and provides quality control on clinical trial data.   The CRC also hosts the Molecular Epidemiology Resource.  The CRC provides a critical link between clinical research and the specific projects and cores. The CRC is co-directed by Brian Link, M.D., at the HCCC and Thomas M. Habermann, M.D., at the MCCC.  Other key members of the CRC are Protocol Development Coordinators (PDC), Clinical Research Associates (CRA), and Research Assistants (RA) located at both sites. The PDC functions with the PI and IRB to develop and activate the clinical trial and consent form. The CRA and RA assists in recruiting patients to the trials, schedules protocol tests, and collaborates with the Biospecimens Core and the specific research projects to ensure that translational research samples from patients are obtained according to protocol guidelines. The CRA and RA arranges for data entry into the Biostatistics and Bioinformatics Core.  The CRC has been very active and developed 4 clinical trials that accrued 113 patients. In addition, the Molecular Epidemiology Resource accrued 1,331 eligible patients through December 31, 2005.  The CRC has worked with the PIs to develop the 4 projects in this competitive renewal and is in the process of protocol development for clinical trials outlined in Projects 1, 2, and 4. In addition, the CRC will provide ongoing support for the Molecular Epidemiology Resource that is being used in Project 3 and a developmental project, and is expected to be an increasingly valuable resource as it matures.  The CRC will continue to recruit patients to the 4 trials that remain active from the first funding period and follow patients already enrolled until protocol endpoints are met.  The CRC is also responsible for the clinical trials monitoring plans, which have been fully developed at both sites.  These monitoring plans assure that the appropriate expertise is available to review the patient protocols, obtain IRB approval, and provide periodic review of ongoing protocol review to maximize patient safety. In summary, the CRC has been an extremely valuable resource during the first funding period.  It has functioned at a high level and will be a very important core in the next funding period to support the clinical trials research, translational research projects, and epidemiology projects.

Project 1: A Novel Approach to the Immunotherapy of B Cell Malignancy

Project 2: Signal Transduction Inhibitor Therapy for Lymphoma

Project 3: Biology and Epidemiology of APRIL and Blys in B-cell and NHL

Project 4: Regulatory T-Cells in the Tumor Microenvironment of B-Cell Non-Hodgkin Lymphoma

Core 1:  Administration

Core 2: Biospecimens

Core 3: Biostatistics and Bioinformatics

Core 4: Clinical Research