Currently, the Hematologic Malignancies Program is active in a wide range of research projects, from basic molecular studies to investigations into environmental factors related to the incidence of blood cancers. They are working to uncover the causes of best treatments for diseases such as Hodgkin and non-Hodgkin lymphoma, multiple myeloma, myelofibrosis, and B cell chronic lymphocytic leukemia. Some of the ongoing research endeavors include:
Lenalidomide for Multiple Myeloma
Mayo researchers led the first published Phase II trial to demonstrate the activity of lenalidomide, a novel immunomodulatory analogue of thalidomide, in multiple myeloma (Rajkumar, 2005; Lacy, 2007). This trial was also the first to show a remarkably high response rate and overall survival in newly diagnosed multiple myeloma patients with lenalidomide-based therapy.
The investigators followed the success of the Phase II study by conducting the first randomized trial of lenalidomide in newly diagnosed multiple myeloma (E4A03). This trial accrued 445 patients in just over one year and found that the overall survival with lenalidomide plus low-dose dexamethasone (Dex) was significantly superior to lenalidomide plus high-dose Dex with a one-year survivals of 96 percent versus 87 percent, respectively (Rajkumar, 2007). This study has major implications for the treatment of multiple myeloma since the one- and two-year survival rate observed in the lenalidomide low-dose Dex arm of this trial are the highest reported in any Phase III trial in multiple myeloma to date. The trial was a landmark trial that also changed the standard of care in multiple myeloma by demonstrating that the standard high dose of corticosteroids used in the treatment of multiple myeloma shortened overall survival compared to lower doses.
Mayo researchers are now testing the addition of cyclophosphamide to lenalidomide and dexamethasone (CRD) in a Phase II clinical trial. Hematologic Malignancies Program investigators are also leading the next generation of multiple myeloma trials with novel three- (lenalidomide-bortezomib-dexamethasone) and four-(cyclophosphamide-lenalidomide-bortezomib-dexamethasone) drug combination multi-center randomized trials. The Program was also the first group in the United States to develop the combination of lenalidomide with melphalan and prednisone (MPR) for newly diagnosed patients with multiple myeloma who are not transplant eligible. This has now led to the development of a national Phase III clinical trial by ECOG (E1A06) led by Keith Stewart, M.B.Ch.B.
Proteasome Inhibitors for Multiple Myeloma
Program researchers have also been engaged in the development of new proteasome inhibitor-based strategies for the treatment of multiple myeloma. Dr. Stewart presented the preliminary results of the Phase I study of the novel proteasome inhibitor carfilzomib at the 2007 American Society of Clinical Oncology (ASCO) meeting. This agent has shown clear evidence of anti-tumor activity in the Phase I clinical trial, the Phase II clinical trial is accruing and Dr. Stewart will be the principle investigator on the pivotal Phase III clinical trial.
Mayo investigators have also developed trials of innovative combinations, such as a Phase II trial of bortezomib in combination with dexamethasone and cyclophosphamide, which produced an unprecedented 100 percent overall response rates with over 50 percent of patients attaining a clinical response (as reported by Craig Reeder, M.D., at the American Society of Hematology 2007 meeting). Translational research studies are coupled with these clinical trials. Functional genetic studies using high throughput siRNA to search for genes, that when down-regulated, enhance susceptibility to bortezomib have been performed using clinical patient samples. Using this strategy, the researchers identified that down regulation of the aurora kinase A and B acts synergistically with bortezomib inhibition. These results provide the rationale for two Phase I clinical trials with two small molecule inhibitors of aurora kinases - currently in development.
Lymphoma - Molecular and Cellular Biology
Basic studies using Mayo Clinic Cancer Center and Lymphoma SPORE resources were focused on three areas - the tumor necrosis factor (TNF) family member BLyS, or B-Lymphocyte Stimulator (Novak, 2006); the role of regulatory T-cells (Tregs); and mTOR inhibition. The BLyS work is being pursued further in the SPORE in collaboration with James Cerhan, M.D., Ph.D., co-leader of the Genetic Epidemiology and Risk Assessment Program.
Stephen Ansell, M.D., Ph.D., and his group, continues to investigate the role of Tregs in lymphoma. Prior work identified a subset of CD4(+)CD25(+) T cells in fresh human lymphoma samples with high levels of CTLA-4 and Foxp3 - intratumoral Treg cells. There were higher numbers of Tregs in lymphoma samples compared to reactive nodes. Functional studies demonstrated that these CD4(+)CD25(+) T cells suppressed the proliferation and cytokine (IFN-γ multiple myeloma and IL-4) production of infiltrating CD4(+)CD25(-) T cells in response to PHA stimulation. The malignant B-cells were shown to induce the recruitment of Tregs into the nodal microenvironment (Yang, et al. Cancer Research, 2006). A second study (Yang et al. Blood, 2006) furthered our understanding of Tregs in non-Hodgkin lymphoma by finding that intratumoral Treg cells inhibit the proliferation and granule production of activated autologous infiltrating CD8(+) T cell. In addition, degranulation and subsequent cytotoxic activity of infiltrating CD8(+) T cells exposed to lymphoma B cells was completely attenuated by the presence of intratumoral T(reg) cells. A third study (Yang et al. Blood, 2007) demonstrated that CD4+ CD25- cells in malignant lymphoma nodes could express FOXP3 and function as Tregs. CD70 expression on lymphoma cells contributed to the activation of Tregs and blockade of CD27:CD70 interaction with anti-CD70 MoAb was successful at blocking the induction of Tregs. Taken together, these data indicate that human lymphoma B cells are sensitive to autologous CTL-mediated cell death but are protected by the inhibitory function of intratumoral T(reg) cells. The group has initiated a trial in the North Central Cancer Treatment Group (NCCTG) to treat both the malignant B-cells and the Tregs based on these laboratory studies. The NCCTG is testing in a Phase II trial a combination of rituximab to target the malignant B-cell and a denileukin diftitox (Ontak) to target the T regulatory cells.
Thomas Witzig, M.D.; Scott Kaufmann, M.D., Ph.D.; and Shaji Kumar, M.D.; have developed a laboratory program to evaluate the effect of mTOR inhibitors as single-agents and in combination with other agents for treatment of lymphoma.
In the area of biomarkers, the group continued to investigate the importance of baseline absolute lymphocyte count on response and survival in non-Hodgkin lymphoma. The Lymphoma group hypothesized that a stronger patient immune system prior to rituximab therapy for follicular (grades 1 and 2) lymphomas would result in better response rates and longer time-to-progression. A recent study (Behl et al. British Journal of Haematology, 2007) of 79 follicular lymphoma patients treated with single agent rituximab demonstrated a superior time-to-progression in those patients with an absolute lymphocytic count greater than or equal to 0.89 x 10(9)/liters (n = 40) compared with those with an absolute lymphocytic count less than 0.89 x 10(9)/liters (n = 39) at the time of rituximab therapy (median: 36.5 vs. 8.1 months, respectively, P < 0.0009). Higher complete response rates were observed in patients with the higher count (23/40, 58 percent; compared with 5/39, 13 percent; P < 0.0001). Multivariate analysis showed absolute lymphocytic count to be an independent predictor for time-to-progression. The importance of absolute lymphocytic count recovery after transplant is being tested in a new Phase III trial conducted by Luis Porrata, M.D., and the transplant group to learn whether increased harvest of lymphocytes pre-transplant can increase the absolute lymphocytic count recovery.
Chronic Lymphocytic Leukemia
Efforts in the epidemiology of chronic lymphocytic leukemia (CLL) are led by Susan Slager, Ph.D., and James Cerhan, M.D., Ph.D. Dr. Slager leads a familial CLL research consortium (U01 award) involving investigators from seven institutions. The overall aim of this research is to identify genetic risks of CLL through the evaluation of high-risk CLL families. Dr. Cerhan leads R01-funded research investigating the genetic and environmental risk of CLL using an association study of unrelated cases and controls. These two studies are complementary and are building a rich epidemiological resource that will be positioned to identify and characterize the risk of CLL.
Asish Ghosh, Ph.D., working with Neil Kay, M.D., and other colleagues, has demonstrated that microvesicles circulate in the plasma of CLL patients. These microvesicles are generated from either lymphocytes or platelets and importantly contain multiple proteins, receptors and cytokines capable of interaction with CLL B cells or the stromal microenvironment. These findings represent a novel means of cell-to-cell communication and offer unique opportunities for therapy in CLL.
In the same laboratory, Wei Ding, M.D., Ph.D., developed a unique bone biopsy-derived stromal cell system that permits analysis of CLL B cell stromal cell crosstalk. Using this stromal cell system she demonstrated that both AKT and MAPK are activated on stromal cells following co-culture with CLL B cells. This work builds on our long-term interest in cell-to-cell interaction in the microenvironment of CLL patients and in particular promises to allow for further dissection of the angiogenic switch that occurs when CLL B cells reside on stromal cells (Kay et al. Leukemia Research, 2007).
The CLL group is also working with our basic science groups in Mayo Clinic Cancer Center. For example, Dr Kay has recently collaborated with Daniel Billadeau, Ph.D. In this joint project, they demonstrated that the inhibition of glycogen synthase kinase-3 beta led to a decrease in NF-κB-mediated gene transcription (Ougolkov et al. Blood, 2007). The inhibition of GSK-3 inhibits NF-κB binding to its target gene promoters through an epigenetic mechanism thus enhancing apoptosis in CLL B cells ex vivo. GSK-3 is a potential therapeutic target for future therapeutic studies in CLL. The group showed that the multi-kinase inhibitor sorafenib interacts synergistically with the proteasome inhibitor bortezomib (Yu et al. Molecular Cancer Therapeutics, 2006). This finding provides sound rationale for future clinical trials in CLL with this unique combination approach.
In collaboration with the Lymphoma SPORE, the CLL group published the results of a new biomarker for CLL, the serum free light chain (SFLC) assay (Martin et al. Translational Research, 2007). Forty-four percent (8/18) of patients with CLL had a monoclonal protein detected by SFLC and six cases were detected only by the SFLC method; therefore, SFLC offers another method of detecting minimal residual disease in patients with CLL. In other analyses, Hematologic Malignancies Program researchers have begun to analyze the distinction between CLL and the recently characterized condition of monoclonal B-cell lymphocytosis (Maddocks-Christianson et al. British Journal of Haematology, 2007). In a recent publication by Mukherjee et al, samples from seven patients with CLL were treated in vitro with gold nanoparticles that were linked to antibody to VEGF. The induction of apoptosis with the gold nanoparticles linked to the anti VEGF was significantly higher than the CLL cells exposed to anti VEGF or gold nanoparticles alone. This provides proof of concept for the potential of nanogold-based therapies in CLL.
Chronic Myeloid Disorders
In addition to the research being conducted at Mayo Clinic, Hematologic Malignancies Program investigators are collaborating with other scientists in the United States and the United Kingdom on the discovery of disease genes, detecting mutant proteins in blood and tissue that predispose some people to these diseases, and developing dendritic cell and other specially targeted therapies for treatment.
In the last funding period the Myeloid group made significant contributions to the understanding of the pathophysiology of the myeloproliferative disorders. In 2006, Pardanani et al described the mutational frequency, disease specificity, and clinical correlates, using genomic DNA from 1,182 patients with myeloproliferative and other myeloid disorders and 64 healthy controls. The DNA was screened for MPL515 mutations, regardless of JAK2V617F mutational status. In addition to testing for MPLW515L, they discovered a new mutation (MPLW515K). The MPLW515L or MPLW515K mutations were detected in patients with myeloid metaplasia or essential thrombocythemia at a frequency of approximately five percent and one percent and could be observed concurrently. They were not observed in patients with polycythemia vera (PV) or other myeloid disorders. Three patients with myelofibrosis with concurrent MPLW515L/K and JAK2V617F were studied at 25 time points over a period of 4 to 8 years and it was found that allele burden fluctuated, but remained high for MPLW515L/K and low for JAK2V617F suggesting that both mutations are early events in myelofibrosis and that allele burden, rather than the mere presence of these mutations, might be relevant to phenotypic variation in myeloproliferative disorders (Lasho et al. British Journal of Haematology, 2006). Animesh Pardanani, M.D., also demonstrated that MPLW515K, but not JAK2V617F, occurs in vitro expanded CD4+ lymphocytes suggesting that MPLW515K either provide a greater growth advantage to lymphoid-lineage cells or occur in a more primitive lympho-myeloid progenitor (Pardanani et al. Leukemia, 2007). Single hematopoietic colony analysis of patients with JAK2V617F and MPLW515L/K mutations demonstrated clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell (Pardanani et al. Stem Cells, 2007).
Ayalew Tefferi, M.D., and colleagues had previously shown that homozygosity for the JAK2V617F mutation in the granulocytes of patients with polycythemia vera was associated with a higher hemoglobin level, increased incidence of pruritus, a higher rate of fibrotic transformation and higher PRV-1 transcript level in blood granulocytes (Tefferi, et al. British Journal of Haematology, 2006). Studies in 2007 did not confirm that bone marrow allele burden in polycythemia vera correlated with hemoglobin level, platelet count, thrombosis or age, but did correlate with leukocytosis, pruritus and microvascular symptoms and pointed out the potential confounding effect of sample accrual time on result interpretation (Tefferi et al. European Journal of Haematology, 2007). Dr. Pardanani also assessed the prevalence of JAK2 exon 12 mutations in six cases of JAK2V617F-negative polycythemia vera and found mutations in five patients (Pardanani et al. Leukemia, 2007).
In the myelodysplastic syndromes, David Steensma, M.D., confirmed the presence of JAK2V617F in patients with refractory anemia with ringed sideroblasts with associated with marked thrombocytosis (RARS-T) in two of three patients with this disorder and highlighted its rarity amongst over 1,500 patients with myelodysplastic syndromes seen at Mayo Clinic over a six-year period (Steensma et al. Haematologica, 2006). He also demonstrated that acquired mutations in inherited sideroblastic anemias are uncommon in refractory anemia with ringed sideroblasts (Steensma et al. Leukemia Research, 2007).
Dr. Pardanani and colleagues have worked with TargeGen Inc. to perform preclinical testing of TG101209, an orally bioavailable small molecule that potently inhibits JAK2, FLT3 and RET. TG101209 was shown to inhibit the growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations. Therapeutic efficacy was demonstrated in a nude mouse model (Pardanani et al. Leukemia, 2007). Testing of other JAK2V617F inhibitors is in progress and the clinical trial of TG101209 is beginning.
The amyloidosis program at Mayo has expanded from the clinical area to the laboratory; even though it is not yet recognized as a critical area of research at the National Institutes of Health. Rafael Fonseca, M.D., and colleagues, is concentrating on the genetic and cytogenetic nature of the clonal cells of the plasma cell disorders. To achieve this, they use a variety of tools: molecular genetics, FISH, PCR, arrays, etc. The Fonseca Laboratory currently has five full time technologists and three post-doctoral fellows.
Non-Hodgkin Lymphoma (NHL) incidence and mortality rates have increased over the past 50 years, and these trends are largely unexplained. The five-year survival rates were approximately 50 percent during most of this time, and have only recently begun to improve. At present, we cannot accurately predict who will and will not do well after their NHL diagnosis. This study will ask the question of whether survival is in part determined by background genetic variation in immune function and metabolism of agents used to treat lymphoma patients. Mayo researchers are hopeful that identifying these genetic patterns will accomplish two goals: 1) help physicians decide which treatments a patient is most likely to respond to best, with minimal side effects; and 2) help researchers identify new drug targets for treatment.
In Project #5, Mayo investigators proposed to systematically test hypotheses that genetic differences (polymorphisms) related to immune function and regulation and to the metabolism and function of specific anti-cancer drugs may affect survival rates for NHL and a closely related cancer called chronic lymphocytic leukemia (CLL).
Specific aims were:
To test these hypotheses, the project is enrolling all newly diagnosed NHL and CLL patients seen at Mayo Clinic Rochester or University of Iowa within nine months of their diagnosis. The researchers are collecting blood (for DNA), treatment data, and other clinical and laboratory factors, and are following all patients after diagnosis in order to identify clinical outcomes. Genetic variability (polymorphisms) within the selected genes will be identified using the Mayo Clinic Cancer Center Genotyping Shared Resource. The results of the genotyping are then linked to treatment and outcome data to see if we can predict who has a better outcome after accounting for disease and treatment differences. The researchers completed the first round of genotyping in 2007, and should have early results in mid 2008.
As part of the SPORE renewal in 2007, this project became the Molecular Epidemiology Resource (MER), and is a core resource available to SPORE researchers. Through 2007, 2,450 participants have enrolled into the MER, 1,842 from Mayo Clinic and 608 from the University of Iowa. Some of the participants will be reaching their six-year follow-up in the summer of 2008.
The three most recent grants funded that use the MER:
Biology and Epidemiology of BLyS and APRIL in Follicular Lymphoma
Genetic Predictors of Prognosis in Mantle Cell Lymphoma
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