Daphne R. Friedman

Patterns of microRNA expression characterize stages of human B-cell differentiation

Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Chronic lymphocytic leukemia and regulatory B cells share IL-10 competence and immunosuppressive function

Chronic lymphocytic leukemia (CLL) can be immunosuppressive in humans and mice, and CLL cells share multiple phenotypic markers with regulatory B cells that are competent to produce interleukin (IL)-10 (B10 cells). To identify functional links between CLL cells and regulatory B10 cells, the phenotypes and abilities of leukemia cells from 93 patients with overt CLL to express IL-10 were assessed. CD5+ CLL cells purified from 90% of the patients were IL-10-competent and secreted IL-10 following appropriate ex vivo stimulation. Serum IL-10 levels were also significantly elevated in CLL patients. IL-10-competent cell frequencies were higher among CLLs with IgVH mutations, and correlated positively with TCL1 expression. In the TCL1-transgenic (TCL1-Tg) mouse model of CLL, IL-10-competent B cells with the cell surface phenotype of B10 cells expanded significantly with age, preceding the development of overt, CLL-like leukemia. Malignant CLL cells in TCL1-Tg mice also shared immunoregulatory functions with mouse and human B10 cells. Serum IL-10 levels varied in TCL1-Tg mice, but in vivo low-dose lipopolysaccharide treatment induced IL-10 expression in CLL cells and high levels of serum IL-10. Thus, malignant IL-10-competent CLL cells exhibit regulatory functions comparable to normal B10 cells that may contribute to the immunosuppression observed in patients and TCL1-Tg mice.

SET oncoprotein overexpression in B-cell chronic lymphocytic leukemia and non-Hodgkin lymphoma: a predictor of aggressive disease and a new treatment target

B-cell chronic lymphocytic leukemia (CLL), an incurable leukemia, is characterized by defective apoptosis. We found that the SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A) tumor suppressor, is overexpressed in primary CLL cells and B-cell non-Hodgkin lymphoma (NHL) cell line cells. In CLL, increased levels of SET correlated significantly with disease severity (shorter time to treatment and overall survival). We developed SET antagonist peptides that bound SET, increased cellular PP2A activity, decreased Mcl-1 expression, and displayed selective cytotoxicity for CLL and NHL cells in vitro. In addition, shRNA for SET was cytotoxic for NHL cells in vitro. The SET antagonist peptide COG449 inhibited growth of NHL tumor xenografts in mice. These data demonstrate that SET is a new treatment target in B-cell malignancies and that SET antagonists represent novel agents for treatment of CLL and NHL.

Cocaine-induced pseudovasculitis.

Pseudovasculitis is a disease process that mimics the presentation and possibly the laboratory findings of true vasculitis. However, biopsy specimens do not reveal the typical histopathologic findings expected in vasculitis. One often overlooked cause of pseudovasculitis is cocaine use, which has been described in case reports to cause aggressive nasal destruction and various skin lesions and thus has been confused with Wegener granulomatosis or leukocytoclastic vasculitis. Unfortunately, serologic tests such as antinuclear antibody or antineutrophil cytoplasmic antibody cannot reliably differentiate between these entities. We describe a patient who presented with what was believed to be Wegener granulomatosis affecting the skin and upper airway. However, findings from repeated biopsies did not support this diagnosis, and the only unifying diagnosis was cocaine-induced pseudovasculitis. The ability to recognize and differentiate between true vasculitis and pseudovasculitis is essential for the clinician because treatment options are radically disparate.

A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia

Purpose: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by a variable clinical course. Several parameters have prognostic capabilities but are associated with altered response to therapy in only a small subset of patients. Experimental Design: We used gene expression profiling methods to generate predictors of therapy response and prognosis. Genomic signatures that reflect progressive disease and responses to chemotherapy or chemoimmunotherapy were created using cancer cell lines and patient leukemia cell samples. We validated and applied these three signatures to independent clinical data from four cohorts, representing a total of 301 CLL patients. Results: A genomic signature of prognosis created from patient leukemic cell gene expression data coupled with clinical parameters significantly differentiated patients with stable disease from those with progressive disease in the training data set. The progression signature was validated in two independent data sets, showing a capacity to accurately identify patients at risk for progressive disease. In addition, genomic signatures that predict response to chlorambucil or pentostatin, cyclophosphamide, and rituximab were generated and could accurately distinguish responding and nonresponding CLL patients. Conclusions: Thus, microarray analysis of CLL lymphocytes can be used to refine prognosis and predict response to different therapies. These results have implications for standard and investigational therapeutics in CLL patients. (Clin Cancer Res 2009;15(22):694755)

Informational needs assessment of non‐Hodgkin lymphoma survivors and their physicians

Cancer Survivorship Care Plans (SCPs) are a recommended part of medical care for cancer survivors. We sought to identify the medical and psychosocial informational needs of non-Hodgkin lymphoma survivors and their physicians in cancer survivorship care to be included in SCPs. Questionnaires were mailed to eligible lymphoma survivors and their physicians, querying their preferences about informational needs in SCPs. The survivor cohort had a median age at diagnosis of 62, with 57% Female, 87% White, and 76% from North Carolina. The physician cohort was comprised of oncologists (27%) and nononcologists (73%), and 86% practiced in North Carolina. Greater than 60% of both survivors and physicians preferred an oncologist and primary care provider comanage cancer survivorship care. The most highly rated informational needs were medical issues, although there were differences between survivors and physicians for many of the informational needs queried. There was higher concordance between patient and physician responses for medical issues (63 to 100%) compared to psychosocial issues (25 to 63%). Important components of SCPs for lymphoma survivors and their physicians may go unrecognized. Tailoring SCPs to patient or physician users may better accommodate the differences in need for particular cancer survivorship care information.

Perifosine treatment in chronic lymphocytic leukemia: results of a phase II clinical trial and in vitro studies

Abstract Because of the importance of the phosphoinositide 3-kinase (PI3K)/AKT pathway in chronic lymphocytic leukemia (CLL), we evaluated in vitro cytotoxicity induced by perifosine, an AKT inhibitor, in CLL lymphocytes and found that the mean 50% effective dose (ED50) was 313 nM. We then performed a phase II trial of perifosine in patients with relapsed/refractory CLL to assess response, outcomes, toxicity and ex vivo correlative measures. After 3 months of treatment, six of eight patients showed stable disease, one achieved a partial response and one had progressive disease. Median event-free survival and overall survival in all patients treated were 3.9 and 9.7 months. Adverse events included hematologic, infectious/fever, pain, gastrointestinal and constitutional toxicities. Unexpectedly, AKT phosphorylation in CLL lymphocytes from treated patients was not correlated with response. Additionally, perifosine did not inhibit AKT phosphorylation in cultured CLL lymphocytes. Perifosine is cytotoxic to CLL cells in vitro, and largely induces stabilized disease in vivo, with an AKT-independent mechanism.

Phase II study of cenersen, an antisense inhibitor of p53, in combination with fludarabine, cyclophosphamide and rituximab for high-risk chronic lymphocytic leukemia.

Abstract Patients with chronic lymphocytic leukemia (CLL) with deletion or mutation of TP53 have exceedingly poor clinical outcomes. Cenersen, an oligonucleotide targeting TP53, has been shown to abrogate the activity of TP53 gain-of-function mutants and to increase sensitivity of lymphoma cells to cytotoxic chemotherapy in vitro. We combined cenersen with fludarabine, cyclophosphamide and rituximab (FCR) as treatment for patients with high-risk CLL. The purpose of this phase II study was to determine the overall response rate, response duration and toxicity of cenersen administered in combination with FCR. Twenty patients with relapsed or high-risk CLL were evaluated. Nineteen patients were previously treated. The complete response rate was 18%; the overall response rate was 53%. Median progression-free and overall survival was 5.3 and 10.6 months, respectively. The most common serious adverse events were neutropenia and thrombocytopenia. In this single arm phase II study, cenersen combined with FCR yielded clinical responses with acceptable toxicity in patients with high-risk CLL.

Statin use and need for therapy in chronic lymphocytic leukemia

The in vitro observation that statins reduce CD20 surface expression on B lymphocytes, induce conformational changes in CD20, and decrease the ability of rituximab to bind to B-cell lymphoma cells raised the question of whether concomitant use of these agents with rituximab-containing chemotherapy regimens might reduce these regimens’ effectiveness [1]. A retrospective analysis of patients with follicular and diffuse large B-cell lymphomas treated with rituximab-containing regimens did not demonstrate decreased response to these regimens among patients who received statin drugs at the time of therapy. However, patients with follicular lymphoma who took statin drugs at the time of diagnosis or therapy had an improved event-free survival [2]. Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy that is similar to follicular lymphoma in several ways: both are indolent B-cell malignancies, often with long latencies, in which therapy is almost never curative. Because of the findings in follicular lymphoma, we considered whether statins might have an effect in CLL. At a pre-clinical level, there is already in vitro evidence that statins have anti-CLL activity, inducing CLL cytotoxicity and apoptosis [3,4]. Statins inhibit 3hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, reducing circulating total cholesterol and low-density lipoprotein levels [5]. Lipoproteins and associated enzymes have prognostic significance in CLL. For example, elevated CLL cell lipoprotein lipase (LPL) is associated with poor prognosis [6,7]. Apolipoprotein E (APOE) genotype has prognostic significance in American, British, and Italian patients with CLL [6,8,9]. Recently, using a metabolomic approach, significantly higher serum cholesterol and fatty-acid side chains from very-low-density lipoproteins and low-density lipoproteins were found in high-risk patients with CLL [10]. Despite these findings, one retrospective study of patients with earlystage CLL who took statins at the time of diagnosis found no impact on clinical outcomes [11]. Because of the data regarding statin use in follicular lymphoma as well as the correlation between lipoprotein pathways and prognosis in CLL, we hypothesized that statin use in our cohort of patients with CLL might affect the need for treatment and the time to first treatment. Patients with CLL were enrolled in an Institutional Review Board-approved study at the Duke University and Durham VA Medical Centers. Clinical data were collected from chart review according to the National Cancer Institute (NCI) Working Group criteria [12]. Follow-up time was calculated from the time of diagnosis to the last documented evaluation at our Centers. Statin use at diagnosis and at treatment was determined from chart review, within 3 months before to 3 months after each time point. Data regarding statin use, including dose and type, as well as total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides, were collected. Prognostic markers, including IgVH (immunoglobulin heavy chain variable) mutational status, CD38, ZAP-70, and LPL expression,