David Spaner

Clinical Efficacy of Lenalidomide in Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia: Results of a Phase II Study

PURPOSEnPatients with relapsed or refractory chronic lymphocytic leukemia (CLL) have profound immune defects and limited treatment options. Given the dramatic activity of lenalidomide in other B-cell malignancies and its pleotropic immunomodulatory effects, we conducted a phase II trial of this agent in CLL.nnnPATIENTS AND METHODSnPatients with relapsed or refractory B-cell CLL (B-CLL) were eligible if they required treatment as per the National Cancer Institute Working Group 1996 guidelines. Lenalidomide was administered orally at 25 mg on days 1 through 21 of a 28-day cycle. Response was assessed after each cycle. Patients were to continue treatment until disease progression, unacceptable toxicity, or complete remission. Rituximab was added to lenalidomide on disease progression.nnnRESULTSnForty-five patients were enrolled, with a median age of 64 years. Sixty-four percent of the patients had Rai stage III or IV disease, and 51% were refractory to fludarabine. The overall response rate was 47%, with 9% of the patients attaining a complete remission. Fatigue, thrombocytopenia, and neutropenia were the most common adverse effects noted in 83%, 78%, and 78% of the patients, respectively.nnnCONCLUSIONnLenalidomide is clinically active in patients with relapsed or refractory B-CLL. These findings are encouraging and warrant further investigation of this agent in the treatment of this disorder.

The miR-17-92 cluster expands multipotent hematopoietic progenitors whereas imbalanced expression of its individual oncogenic miRNAs promotes leukemia in mice

The miR-17-92 cluster and its 6 encoded miRNAs are frequently amplified and aberrantly expressed in various malignancies. This study demonstrates that retroviral-mediated miR-17-92 overexpression promotes expansion of multipotent hematopoietic progenitors in mice. Cell lines derived from these miR-17-92-overexpressing mice are capable of myeloid and lymphoid lineage differentiation, and recapitulate the normal lymphoid phenotype when transplanted to nonobese diabetic/severe combined immunodeficiency mice. However, overexpression of individual miRNAs from this locus, miR-19a or miR-92a, results in B-cell hyperplasia and erythroleukemia, respectively. Coexpression of another member of this cluster miR-17, with miR-92a, abrogates miR-92a-induced erythroleukemogenesis. Accordingly, we identified several novel miR-92a and miR-17 target genes regulating erythroid survival and proliferation, including p53. Expression of this critical target results in marked growth inhibition of miR-92a erythroleukemic cells. In both murine and human leukemias, p53 inactivation contributed to the selective overexpression of oncogenic miR-92a and miR-19a, and down-regulation of tumor-suppressive miR-17. This miR-17-92 expression signature was also detected in p53- B-cell chronic lymphocytic leukemia patients displaying an aggressive clinical phenotype. These results revealed that imbalanced miR-17-92 expression, also mediated by p53, directly transforms the hematopoietic compartment. Thus examination of such miRNA expression signatures should aid in the diagnosis and treatment of cancers displaying miR-17-92 gene amplification.

The inositol phosphatase SHIP-1 is negatively regulated by Fli-1 and its loss accelerates leukemogenesis

The activation of Fli-1, an Ets transcription factor, is the critical genetic event in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. Fli-1 overexpression leads to erythropoietin-dependent erythroblast proliferation, enhanced survival, and inhibition of terminal differentiation, through activation of the Ras pathway. However, the mechanism by which Fli-1 activates this signal transduction pathway has yet to be identified. Down-regulation of the Src homology 2 (SH2) domain-containing inositol-5-phosphatase-1 (SHIP-1) is associated with erythropoietin-stimulated erythroleukemic cells and correlates with increased proliferation of transformed cells. In this study, we have shown that F-MuLV-infected SHIP-1 knockout mice display accelerated erythroleukemia progression. In addition, RNA interference (RNAi)-mediated suppression of SHIP-1 in erythroleukemia cells activates the phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways, blocks erythroid differentiation, accelerates erythropoietin-induced proliferation, and leads to PI 3-K-dependent Fli-1 up-regulation. Chromatin immunoprecipitation and luciferase assays confirmed that Fli-1 binds directly to an Ets DNA binding site within the SHIP-1 promoter and suppresses SHIP-1 transcription. These data provide evidence to suggest that SHIP-1 is a direct Fli-1 target, SHIP-1 and Fli-1 regulate each other in a negative feedback loop, and the suppression of SHIP-1 by Fli-1 plays an important role in the transformation of erythroid progenitors by F-MuLV.

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage‐dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell‐cell interaction, and cell spreading. Treatment with EGF increased cell adhesion‐regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion‐regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle‐associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF‐induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion. J. Cell. Biochem. 77:569–583, 2000.

A phase I/II trial of oxidized autologous tumor vaccines during the “watch and wait” phase of chronic lymphocytic leukemia

Based on their activity in patients with advanced stage chronic lymphocytic leukemia (CLL), a phase I/II study was designed to evaluate the feasibility, safety, and efficacy of autologous vaccines made from oxidized tumor cells in patients with earlier stage CLL, and to determine an optimal schedule of injections. Eighteen patients (at risk for disease progression and with white blood cell counts between 15 and 100×106xa0cells/ml) were injected intramuscularly with 10xa0ml of oxidized autologous blood (composed mainly of CLL cells) either 12 times over 6xa0weeks (group 1), 12 times over 16xa0days (group 2), or 4 times over 6xa0weeks (group 3). Fourteen out of eighteen patients had Rai stage 0–II disease, while 4/18 had stage III–IV disease but did not require conventional treatment. Partial clinical responses, associated with enhanced anti-tumor T cell activity in vitro, were observed in 5/18 patients of whom three were in group 2. Stable disease was observed in six patients while disease progression appeared not to be affected in the remaining patients. Toxicity was minimal. Vaccination with oxidized autologous tumor cells appears worthy of further investigation and may be a potential alternative to a “watch and wait” strategy for selected CLL patients.

The scid mouse: mutation in a DNA repair gene creates recipients useful for studies on stem cells, lymphocyte development and graft-versus-host disease.

As described by other contributors to this volume, the scid mouse was initially identified as a mutation affecting the immune system (Bosma et al. 1983). We subsequently showed, however, that this mutation is in a DNA repair gene and that the effects on the immune system are only one of the pleiotropic effects which are manifested in all organ systems (Phillips & Fulop 1989). In this Chapter, we will review the effects of the scid gene on DNA repair, discuss various manifestations of the scid phenotype and comment on how the phenotype is useful for diverse studies of the immune system such as subpopulations of hematopoietic stem cells, the development of B lymphocytes and the leaky scid phenotype, and analysis of cellular interactions in graft-versus-host disease.

Survival of patients with transformed lymphoma in the rituximab era

In the pre-rituximab era, transformation of indolent B-cell lymphoma to diffuse large B-cell lymphoma (DLBCL) was associated with an extremely poor outcome and a median post-transformation survival ranging from 1 to 2xa0years. We evaluated the impact of rituximab-cyclophosphamide, adriamycin, vincristine, prednisone (R-CHOP) on the survival outcomes of transformed lymphoma compared with de novo DLBCL. Between 2002 and 2010, 317 DLBCL patients who were consecutively diagnosed and treated with R-CHOP were identified at our institution. Patients with transformed lymphoma were included if they had not previously received R-CHOP. Patient characteristics, treatment, and outcome data were retrospectively collected. Sixty patients (19xa0%) had transformed lymphoma of which 37 (62xa0%) had transformed from follicular lymphoma, 50 (83xa0%) were chemotherapy naïve, and 58 (96xa0%) were rituximab naïve at the time of treatment. With a median follow-up of 31.4xa0months, 231 patients achieved either complete response or complete response unconfirmed (73xa0%) with no significant difference between de novo DLBCL (nu2009=u2009192, 75xa0%) and the transformed group (nu2009=u200939, 65xa0%) (Pu2009=u20090.25). Six patients (15xa0%) relapsed in the transformed group at a median time to relapse of 29.3xa0months. The 2-year and 5-year overall survivals for all patients were 82 and 72xa0%, respectively. The overall and progression-free survivals for transformed lymphoma and de novo DLBCL were not statistically different (Pu2009=u20090.45 and Pu2009=u20090.38, respectively). With R-CHOP chemotherapy, the prognosis of transformed lymphoma in patients with minimal chemotherapy exposure for indolent disease is similar to that of de novo DLBCL.

Antagonizing Peroxisome Proliferator-Activated Receptor α Activity Selectively Enhances Th1 Immunity in Male Mice.

Females exhibit more robust Th1 responses than males. Our previous work suggested that this sex disparity is a consequence of higher activity of the androgen-induced gene peroxisome proliferator–activated receptor α (PPARα) in male CD4+ T cells. The objective of this study was to elucidate the cellular and molecular mechanism of how PPARα inhibits Th1 responses in male mice. In this study, we found that PPARα functions within CD4+ and CD8+ T lymphocytes and NKT cells to negatively regulate IFN-γ responses in male mice and identified Ifng as the gene target of PPARα repression. Treatment of male CD4+ T cells with the PPARα agonist fenofibrate induced the recruitment of PPARα and the nuclear receptor-interacting protein, nuclear receptor corepressor 1, to specific cis-regulatory elements in the Ifng locus. This recruitment associated with reduced histone acetylation at these sites. Knockdown of nuclear receptor corepressor 1 in primary male T cells abolished the effect of fenofibrate in reducing IFN-γ production. In contrast, treatment of male T cells with IS001, a novel antagonist of PPARα, increased Ifng gene expression and histone acetylation across the Ifng locus. Finally, we investigated the effects of IS001 on IFN-γ responses in mice during infection with the Th1-associated pathogen Listeria monocytogenes and observed that IS001 enhanced IFN-γ production by NKT, CD4+, and CD8+ T cells and improved the survival of male, but not female, mice. Our findings provide a novel mechanism of why IFN-γ responses are more robust in females and introduce a small-molecule IS001 that can be used to enhance Th1 immunity in males.

Prolonged clinical remissions in patients with relapsed or refractory follicular lymphoma treated with autologous stem cell transplantation incorporating rituximab

Three sequential phase II trials were conducted with different immunotherapy approaches to enhance the outcome of autologous transplant (high-dose therapy and autologous stem cell transplantation (HDT/ASCT)) for recurrent follicular lymphoma. Seventy-three patients were enrolled from 1996 to 2009. Patients received HDT/ASCT combined with (1) interferon-α 3xa0MU/m2 subcutaneously (SC) three times per week (TIW) for 2xa0years post-ASCT, (2) rituximab (R) 375xa0mg/m2 for in vivo purging 3–5xa0days pre-stem cell collection and 2u2009×u20094 weekly R at 2 and 6xa0months post-ASCT, respectively, or (3) three infusions of R pre-stem cell collection followed by 6× R weekly and interferon-α 3xa0MU/m2 SC TIW. Although not statistically significant, progression-free survival (PFS) for patients who received rituximab was 56.4 and 49.1xa0% at 5 and 10xa0years compared to 36 and 21xa0% in those who did not receive rituximab. Molecular relapse post-HDT/ASCT was the strongest predictor of PFS in a multivariate analysis. Molecular relapse was coincident with or preceded clinical relapses in 84xa0% of patients who relapsed—median of 12xa0months (range 0–129xa0months). Adverse events included secondary malignancy, transformation to diffuse large B cell lymphoma, prolonged mostly asymptomatic hypogammaglobulinemia, and pulmonary fibrosis. The long-term toxicity profile must be considered when selecting patients for this treatment.

Prolonged molecular and clinical remission after treatment of a patient with follicular lymphoma with rituximab.

Rituximab is a chimeric anti-CD20 monoclonal antibody that has approval for single agent therapy in the treatment of relapsed/refractory low grade or follicular non-Hodgkins Lymphoma. In published phase II trials, molecular remissions of PCR detectable t(14; 18) disease in the peripheral blood have been reported in up to 62% of patients by three months. We report a case of a patient who achieved prolonged clinical and molecular remission following a single four week course of Rituximab that has exceeded any previous remission achieved with cheino-radiotherapy. The implications of molecular remission as a surrogate of clinical remission and molecular relapse as a harbinger of clinical relapse are reviewed and discussed.