Rose C. Beck

Biologic predictors in follicular lymphoma: Importance of markers of immune response

We sought to identify biologic indicators of prognosis in a series of 94 follicular lymphoma (FL) patients, focusing on markers of the host immune response as well as of B-cell maturation. Immune response was assessed with immunostains for CD68 (for lymphoma-associated macrophages, LAMs) and FOXP3 (regulatory T-cells). Lymphoma cells were evaluated for expression of bcl-2, CD10, and MUM-1. Clinical data were obtained for FLIPI, presence of bulky disease, presence of B-symptoms, treatment, and overall survival (OS). For the 69 initially treated patients, extrafollicular CD68+ cells (ef-CD68) and follicular FOXP3+ cells (f-FOXP3) were associated with shorter OS, while receipt of rituximab was associated with longer OS. Multivariable analysis showed ef-CD68 was the only independent factor associated with shorter OS. In subset analysis, ef-CD68 remained statistically significant in rituximab-naïve but not rituximab-treated patients. We confirm the importance of LAMs and f-FOXP3 as predictors of OS in FL.

Automated colorimetric in situ hybridization (CISH) detection of immunoglobulin (Ig) light chain mRNA expression in plasma cell (PC) dyscrasias and non-Hodgkin lymphoma.

Immunohistochemistry (IHC) is frequently used to detect plasma cell (PC) or B cell monoclonality in histologic sections, but its interpretation is often confounded by background staining. We evaluated a new automated method for colorimetric in situ hybridization (CISH) detection of clonality in PC dyscrasias and small B cell lymphomas. Cases of PC dyscrasia included multiple myeloma (MM; 31 cases), plasmacytoma (seven cases), or amyloidosis (one case), while cases of lymphoma included small lymphocytic (three cases), marginal zone (four cases), lymphoplasmacytic (three cases), and mantle cell lymphomas (three cases). Tissue sections were stained for kappa and lambda light chains by IHC and for light chain mRNA by automated CISH using haptenated probes. Twenty-eight of 31 MM cases had detectable light chain restriction by IHC. Thirty of 31 MM cases demonstrated light chain restriction by CISH, including 2 cases with uninterpretable IHC and one case of nonsecretory myeloma, which was negative for light chains by IHC. Seven of 7 plasmacytoma cases had detectable light chain restriction by CISH, including one case of nonsecretory plasmacytoma in which IHC was noninformative. Automated CISH demonstrated monoclonality in 9 of 13 cases of B cell non-Hodgkin lymphoma and had a slightly higher sensitivity than IHC (6 of 13 cases), especially in cases of lymphoplasmacytic and marginal zone lymphoma. Overall, there were no discrepancies in light chain restriction results between IHC, CISH, or serum paraprotein analysis. Automated CISH is useful in detecting light chain expression in paraffin sections and appeared superior to IHC for light chain detection in PC dyscrasias and B cell non-Hodgkin lymphomas, predominantly due to lack of background staining.

The Notch Ligands Jagged2, Delta1, and Delta4 Induce Differentiation and Expansion of Functional Human NK Cells from CD34+ Cord Blood Hematopoietic Progenitor Cells

Notch receptor signaling is required for T cell development, but its role in natural killer (NK) cell development is poorly understood. We compared the ability of the 5 mammalian Notch ligands (Jagged1, Jagged2, Delta1, Delta3, or Delta4) to induce NK cell development from human hematopoietic progenitor cells (HPCs). CD34(+) HPCs were cultured with OP9 stromal cell lines transduced with 1 of the Notch ligands or with OP9 stromal cells alone, in the presence of IL-7, Flt3L, and IL-15. Differentiation and expansion of CD56(+)CD3(-) cells were greatly accelerated in the presence of Jagged2, Delta-1, or Delta-4, versus culture in the absence of ligand or in the presence of Jagged1 or Delta3. At 4 weeks, cultures containing Jagged2, Delta1, or Delta4 contained 80% to 90% NK cells, with the remaining cells being CD33(+) myelogenous cells. Notch-induced NK (N-NK) cells resembled CD56(bright) NK cells in that they were CD16(-), CD94(-), CD117(+), and killer immunoglobulin-like receptors (KIR(-)). They also expressed NKp30, NKp44, NKp46, 2B4, and DNAM-1, with partial expression of NKG2D. The N-NK cells displayed cytotoxic activity against the K562 and RPMI-8226 cell lines, at levels similar to activated peripheral blood (PB) NK cells, although killing of Daudi cells was not present. N-NK cells were also capable of interferon (IFN)-gamma secretion. Thus, Notch ligands have differential ability to induce and expand immature, but functional, NK cells from CD34(+) HPCs. The use of Notch ligands to generate functional NK cells in vitro may be significant for cellular therapy purposes.

Detection of Polyoma Virus in Brain Tissue of Patients with Progressive Multifocal Leukoencephalopathy by Real-Time PCR and Pyrosequencing

We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

Repression of GSK3 restores NK cell cytotoxicity in AML patients

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.

ERK signaling is essential for macrophage development

Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2 flox/flox Lyz2 Cre/Cre mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre mice was enriched for CD11b+ myeloid cells, CD11bhi Gr-1hi neutrophils, Lin- c-Kit+ Sca–1+ hematopoietic stem cells, and Lin- c-Kit+ CD34+ CD16/32+ granulocyte-macrophage progenitors. Culture of bone marrow Lin- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

Aberrant Notch Signaling in the Bone Marrow Microenvironment of Acute Lymphoid Leukemia Suppresses Osteoblast-Mediated Support of Hematopoietic Niche Function

More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-cell acute lymphoblastic leukemia patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biologic impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrated that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution, and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients.

JC virus chromogenic in situ hybridization in brain biopsies from patients with and without PML.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC polyoma virus. Electron microscopy and immunohistochemistry are the traditional methods of confirming the presence of the virus in brain biopsies from these patients. We studied the brain biopsies from 7 patients with PML and 6 patients without PML with chromogenic in situ hybridization (CISH) for the JC polyoma virus using a commercially available probe. The biopsies from the patients with the PML cases were proven to contain the JC polyoma virus by traditional and molecular methods. The CISH findings were compared with the known state of infection. All (7/7) of the biopsies from patients with PML were positive for the presence of polyoma virus by CISH, whereas the biopsies from patients without PML were uniformly negative. CISH seems to be a useful tool for the detection of the JC virus in brain biopsies from patients with PML, and is more accessible because a commercial probe is available.

Production of cytotoxic, KIR‐negative NK cells from CD34+ cord blood cells with the use of Notch signaling

The use of natural killer (NK) cells as cell therapy against acute leukemia is an active area of investigation. The optimal source of cytotoxic NK cells for therapeutic use is presently unknown. With funds from the National Blood Foundation, the authors lab has developed in vitro culture systems that use the Notch receptor ligand Delta4 for the differentiation and expansion of functional NK cells from CD34+ cord blood hematopoietic progenitor cells. These Notch‐induced NK (N‐NK) cells display a predominantly immature, CD56bright surface phenotype, with expression of activating receptors important for leukemia cell recognition and killing, but with an absence of inhibitory receptors that bind major histocompatibility complex (MHC) class I, making them free of restriction by self‐MHC. They are capable of directly killing hematopoietic tumor cell lines and primary leukemia cells in vitro. Thus, cytotoxic, HLA‐independent N‐NK cells may represent a novel cell therapy for hematopoietic malignancy.

Significance of the absolute lymphocyte/monocyte ratio as a prognostic immune biomarker in newly diagnosed multiple myeloma

Significance of the absolute lymphocyte/monocyte ratio as a prognostic immune biomarker in newly diagnosed multiple myeloma