Jeffrey W. Potter

Gene Expression Signatures Predictive of Early Response and Outcome in High-Risk Childhood Acute Lymphoblastic Leukemia: A Children's Oncology Group Study

PURPOSE To identify children with acute lymphoblastic leukemia (ALL) at initial diagnosis who are at risk for inferior response to therapy by using molecular signatures. PATIENTS AND METHODS Gene expression profiles were generated from bone marrow blasts at initial diagnosis from a cohort of 99 children with National Cancer Institute-defined high-risk ALL who were treated uniformly on the Childrens Oncology Group (COG) 1961 study. For prediction of early response, genes that correlated to marrow status on day 7 were identified on a training set and were validated on a test set. An additional signature was correlated with long-term outcome, and the predictive models were validated on three large, independent patient cohorts. Results We identified a 24-probe set signature that was highly predictive of day 7 marrow status on the test set (P = .0061). Pathways were identified that may play a role in early blast regression. We have also identified a 47-probe set signature (which represents 41 unique genes) that was predictive of long-term outcome in our data set as well as three large independent data sets of patients with childhood ALL who were treated on different protocols. However, we did not find sufficient evidence for the added significance of these genes and the derived predictive models when other known prognostic features, such as age, WBC, and karyotype, were included in a multivariate analysis. CONCLUSION Genes and pathways that play a role in early blast regression may identify patients who may be at risk for inferior responses to treatment. A fully validated predictive gene expression signature was defined for high-risk ALL that provided insight into the biologic mechanisms of treatment failure.

Inhibition of β2 Integrin Receptor and Syk Kinase Signaling in Monocytes by the Src Family Kinase Fgr

While beta 2 integrin ligand-receptor recognition interactions are well characterized, less is known about how these events trigger signal transduction cascades to regulate the transition from tethering to firm adhesion, spreading, and transendothelial migration. We have identified critical positive and negative regulatory components of this cascade in monocytes. Whereas the Syk tyrosine kinase is essential for beta 2 integrin signaling and cell spreading, the Src family kinase Fgr is a negative regulator of this pathway. Fgr selectively inhibits beta 2 but not beta 1 integrin signaling and Syk kinase function via a direct association between the Fgr SH2 domain and Syk tyrosine Y342. The inhibitory effects of Fgr are independent of its kinase activity, are dose dependent, and can be overcome by chemokines and inflammatory mediators.

Alterations in neutrophil superoxide production following piroxicam therapy in patients with rheumatoid arthritis

Twenty patients with classical rheumatoid arthritis were enrolled in a study to determine the effects of piroxicam therapy on neutrophil function as defined by chemotaxis and superoxide anion (O2−) production. T-lymphocyte chemotaxis was also evaluated in these patients. Leukocytes were obtained from these subjects initially, after two weeks of placebo treatment, and subsequently after four and 10 weeks of piroxicam therapy (20 mg, once daily). Responses were compared to simultaneously tested normal controls and to the patients own cells obtained at the different time points. Studies showed that four and 10 weeks of piroxicam therapy resulted in significantly suppressed neutrophil O2− production in response to phorbol myristate acetate (PMA) and formyl methionyl leucyl phenylalanine (FMLP). O2− production in response to opsonized zymosan was not significantly affected after four weeks of therapy, but was significantly reduced after 10 weeks of therapy when compared to the patients own cell response after two weeks of placebo treatment. Unlike O2− production, PMN random migration and chemotaxis in response to C5a or FMLP did not differ significantly from normal or untreated patient controls. Analysis of T-lymphocyte migration showed that T-cell random migration or migration to the chemokinetic agent, casein, was not significantly altered by piroxicam therapy. However, when T lymphocytes were tested for chemotaxis in response to lymphocyte-derived chemotactic factor for T cells (LCF), T cell migration was significantly suppressed after 10 weeks of therapy. Furthermore, when T cells from these subjects were cultured for 24 h, random migration was significantly reduced after four and 10 weeks of piroxicam therapy when compared to the patient prior to therapy, and migration in response to LCF was suppressed after four weeks of therapy when compared to normal controls. These data indicate that in patients with rheumatoid arthritis, treatment with piroxicam will significantly suppress PMN O2− production and may also alter the locomotor capacity of T lymphocytes. These actions may contribute to the antiinflammatory effects of piroxicam.

Gene expression overlap affects karyotype prediction in pediatric acute lymphoblastic leukemia.

Gene expression overlap affects karyotype prediction in pediatric acute lymphoblastic leukemia

Human T-lymphocyte chemotactic activity: nature and production in response to antigen

Previous studies have shown that supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells and isolated Leu-2 suppressor/cytotoxic T cells are chemotactic for Leu-3 helper/inducer T cells. The current study shows that lymphocyte chemotactic factor (LCF) is also produced following antigen (tetanus toxoid) challenge of mononuclear cells obtained from recently immunized human donors. LCF was detected in 24-hr supernatants from mononuclear cells challenged with tetanus and was produced maximally at 48 hr. Tetanus toxoid challenge of mononuclear cells obtained from individuals whom had not received a tetanus immunization for 7 to 10 years prior to testing showed little or no production of LCF. Serial studies of these individuals following a tetanus booster immunization showed that LCF was produced by antigen-challenged mononuclear cells obtained 1-5 days postimmunization, was produced optimally 5-15 days postimmunization, and was still produced by antigen-challenged mononuclear cells obtained 6 weeks later. Fractionation of mononuclear cells from immunized donors into glass wool nonadherent lymphocytes, T lymphocytes, and non-T lymphocytes showed that tetanus-toxoid-induced LCF was produced by nonadherent lymphocytes and T cells but not non-T cells. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T-cell subpopulations showed that LCF production by antigen-challenged isolated subpopulations was limited to the Leu-2 suppressor/cytotoxic T-cell subset. Characterization of both Con A and tetanus toxoid-induced LCF by gel filtration on Sephadex G-100 demonstrated the presence of two peaks of LCF corresponding to molecular weights of approximately 14,000-17,000 and 40,000-50,000.

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.

Production and Regulation of Human T Lymphocyte Chemotactic Factor (LCF)

The mobilisation of leukocytes to inflammatory sites is crucial to host defense. Over the years a wealth of information has evolved concerning the mechanism of leukocyte locomotion and factors with the potential specifically to attract these cells to inflammatory foci in vivo. The majority of research in this area has centred on the chemotactic response of neutrophils (PMN) and to a lesser extent on the response of monocytes and macrophages. Various naturally occurring and synthetic chemotactic factors for PMNs and monocytes have been described and they include complement fragments such as C5a (1), products of arachidonic acid metabolism such as leukotriene B4 (2), as well as other cell-derived lipids, such as acetyl glyceryl ether phosphorylcholine (platelet activating factor) (3). More recently, the neurohormones, beta-endorphin, met-enkephalin (4), and substance P (5), have been shown to stimulate mononuclear cell chemotaxis. In many cases, specific receptors for these various chemotactic factors have been defined on both PMNs and monocytes (6–10). In spite of this vast array of information on the mechanism of neutrophil and monocyte locomotion, very little is known about lymphocyte chemotaxis and the agents that are responsible for attracting these cells to sites of antigenic challenge in vivo.