Proteomics-based prediction of clinical response in acute myeloid leukemia.

Objective: Response to chemotherapy is achieved in 60% to 70% of patients with acute myeloid leukemia. The ability to predict responders may help in stratifying patients and exploring different therapeutic approaches for nonresponders. Proteomics methods were used to search for predictive factors or combinations of factors.

Detection of nucleophosmin gene mutations in plasma from patients with acute myeloid leukemia: clinical significance and implications.

Roughly one-third of acute myeloid leukemia (AML) patients exhibit mutations in the nucleophosmin (NPM1) gene, and multiple studies have linked these mutations with a more favorable clinical outcome. We developed an assay for the detection of NPM1 mutations in peripheral blood plasma, and compared the results with clinical outcomes from a single institution. Analyzing plasma from previously untreated AML patients revealed NPM1 insertion mutations in 24 of 98 (24%) patients, with greater sensitivity than existing peripheral blood cell-based tests which showed positivity in only 22 of the 24 patients.

Detection of chromosome translocations by bead-based flow cytometry.

Chromosome translocations resulting in fusion genes have been implicated in leukemogenesis. The paradigm involves the fusion of the genes encoding BCR and ABL, leading to a constitutively active tyrosine kinase. The detection of BCR-ABL has been limited to fluorescence in situ hybridization analysis, reverse transcription-polymerase chain reaction, of mRNA, and Western blot of analysis downstream effectors in the BCR-ABL activated pathway.

Measuring humanized antibodies in plasma of patients treated with antibody-based therapy using bead-based flow cytometry: the story of alemtuzumab.

Alemtuzumab (Campath), the humanized rat monoclonal antibody that targets the CD52 surface antigen, is currently used for treatment of patients with resistant chronic lymphocytic leukemia. Monitoring levels of the antibody in plasma/serum could provide insight into the optimal dosing and scheduling of therapy. Current methods of detecting alemtuzumab in serum or plasma are complicated and difficult to adapt to high-throughput testing.

Cell-free bead-based detection of total and phosphorylated proteins in plasma and cell lysates: detection of FLT3.

Frequently direct measurement of proteins or their phosphorylation in intact cells is not possible, for instance, when cells are too few, frozen, or subject to degradation. We have demonstrated that tumor cells pour their DNA, RNA, and protein content into circulation because of turnover and breakdown of cell structures. Proteins in solution most likely circulate as complexes, which protects them from degradation.

Monitoring cell signaling pathways by quantitative flow cytometry.

As the signaling pathways involved in leukemogenesis are being elucidated, several proteins have emerged as potential targets for therapy. Downstream from those targets are numerous intracellular factors that are constantly modulated. Monitoring those factors could provide insight into the potential efficacy of therapies by predicting which patients will respond to them and by determining the optimal dosage that will inhibit the target protein.

Quantification of surface antigens and quantitative flow cytometry.

Measuring expression levels of cell surface antigens is important for the diagnosis of diseases such as B-cell chronic lymphocytic leukemia and the monitoring of targeted therapy, particularly antibody-based therapy. In some cases, the number of antigens that the therapeutic antibodies bind on the cell surface may reflect the efficacy of therapy. Thus, quantitating the number of molecules on the surface of cells before, during, and after therapy would provide important information for monitoring antibody-based therapy and potentially can be used to adjust dosing.

He use of the antibodies in the diagnosis of leukemia and lymphoma by flow cytometry.

Flow cytometry is an automated analysis of cells passing in the fluid suspension through a laser light beam, which react with monoclonal antibodies specific for a variety of cell surface antigens. A specimen of peripheral blood, bone marrow, or other cell suspension is incubated with fluorescent-labeled antibodies, which bind to target antigens on cell surfaces or--following cell permeabilization--to cytoplasmic and nuclear antigens. The analysis of surface antigens is performed on cells selected (gated) based on light-scatter properties.

Enzymatic activity of circulating proteasomes correlates with clinical behavior in patients with chronic lymphocytic leukemia.

Background: The ubiquitin-proteasome pathway has been implicated in the pathogenesis of many hematologic malignancies.

Methods: The authors measured proteasome peptidase activity levels in plasma samples from 225 patients with chronic lymphocytic leukemia (CLL) and correlated the results with clinical behavior. By using fluorogenic kinetic assays, the enzymatic activity levels of 3 proteasomes were measured: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L).

An immunological method for the detection of BCR-ABL fusion protein and monitoring its activation.

We have developed a simplified sandwich immunoassay to measure free circulating total and phosphorylated fusion BCR-ABL protein in patients with the t(9;22)(q34;q11) chromosomal translocation. The assay is based on immunoprecipitating BCR-ABL protein using beads coated with anti-BCR antibody and detecting the fusion protein with anti-ABL antibody and flow cytometry. We show that this method allows the quantification of this protein in the plasma and may allow the measurement of tumor load.

A potential role for HSP90 inhibitors in the treatment of JAK2 mutant-positive diseases as demonstrated using quantitative flow cytometry.

The V617F mutation of the JAK2 tyrosine kinase is found in a majority of patients with myeloproliferative disorders. Flow cytometry assays for quantitation of phosphorylated and total protein for JAK2, STAT5, and heat shock proteins (HSPs) were developed to facilitate the study of the JAK/STAT pathway. A cell line homozygous for V617F (HEL) was treated with inhibitors of JAK2 tyrosine kinase activity and the HSP90 inhibitor 17-AAG.

Phosphorylation levels of BCR-ABL, CrkL, AKT and STAT5 in imatinib-resistant chronic myeloid leukemia cells implicate alternative pathway usage as a survival strategy.

Ex-vivo studies have suggested that imatinib-resistance in chronic myeloid leukemia (CML) patients occurs despite adequate suppression of BCR-ABL activity. Whether BCR-ABL phosphorylation levels differ between imatinib-sensitive and -resistant patients is not known. We compared the phosphorylation of BCR-ABL in 54 previously untreated CML patients and 62 imatinib-resistant CML patients with progressive disease.

Plasma RNA as an alternative to cells for monitoring molecular response in patients with chronic myeloid leukemia.

Background And Objectives: Quantitation of BCR-ABL mRNA is emerging as the standard of care to monitor the status of chronic myeloid leukemia (CML). Peripheral blood plasma was analyzed in this study because of previous detection of nucleic acids and proteins from tumor cells in plasma samples.

Design And Methods: Reverse transcriptase polyemrase chain reaction was used to establish ratios of BCR-ABL:ABL mRNA in peripheral blood cells and plasma, and absolute levels of BCR-ABL mRNA per unit volume of plasma.

Plasma thrombopoietin compared with immunoglobulin heavy-chain mutation status as a predictor of survival in chronic lymphocytic leukemia.

We investigated the association of plasma thrombopoietin (TPO) and overall survival in 127 patients with previously treated and previously untreated chronic lymphocytic leukemia (CLL). Higher levels of TPO were associated with advanced Rai stage (P < .001), higher levels of beta(2)-microglobulin (beta2-M) (P < .

Clinical relevance of soluble HLA-I and beta2-microglobulin levels in non-Hodgkin's lymphoma and Hodgkin's disease.

Plasma levels of beta-2 microglobulin (beta2M), a subunit of the human leukocyte antigen-class I (HLA-I) molecule, correlate negatively with outcome in non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). We examined the clinical relevance of soluble HLA-I (sHLA-I) levels in NHL and HD. Sera from consecutive NHL (n=65) and HD (n=37) patients were analyzed in a blinded manner.

Proteomic-based prediction of clinical behavior in adult acute lymphoblastic leukemia.

Background: Response in adult acute lymphoblastic leukemia (ALL) can be achieved in a majority of patients. However, unlike pediatric ALL, recurrence is common in adult ALL, and the ability to predict at an early stage which patients are most likely to experience recurrence may help in devising new therapeutic approaches to prevent recurrence.

Methods: Peripheral blood plasma from 57 patients with confirmed ALL was obtained before induction therapy for proteomic analysis.

Angiogenic factors may have a different prognostic role in adult acute lymphoblastic leukemia.

Angiogenesis plays an important role in solid tumors and hematologic malignancies. The prognostic significance of angiogenic factors in adult acute lymphoblastic leukemia (ALL) remains ambiguous. We therefore analyzed the impact of angiogenic factor levels on overall survival of newly diagnosed adult ALL patients.

Alemtuzumab: validation of a sensitive and simple enzyme-linked immunosorbent assay.

Alemtuzumab (MabCampath) is a humanized rat monoclonal antibody that targets the CD52 antigen. It has been approved for the treatment of patients with resistant chronic lymphocytic leukaemia (CLL). Measuring plasma/serum levels of alemtuzumab is important for optimizing the dosing and scheduling of therapy; however, current assays in serum or plasma, based on the capture of alemtuzumab using CD52, are complicated and difficult to adapt for high throughput testing.

Free circulating soluble CD52 as a tumor marker in chronic lymphocytic leukemia and its implication in therapy with anti-CD52 antibodies.

Background: The CD52 antigen is a glycoprotein anchored on the cell membrane of mature B and T lymphocytes, monocytes, and eosinophils. Alemtuzumab (CAMPATH-1H; anti-CD52) is currently approved for the treatment of patients with refractory chronic lymphocytic leukemia (CLL). The authors investigated the possibility that CD52 may be shed from cells and, once soluble, may bind to injected alemtuzumab, forming immune complexes.

Soluble vascular endothelial growth factor receptor 1, and not receptor 2, is an independent prognostic factor in acute myeloid leukemia and myelodysplastic syndromes.

Background: Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are major regulators of angiogenesis, which plays a key role in the growth and dissemination of solid tumors and hematologic neoplasms.

Methods: The authors measured the plasma concentrations of soluble VEGFR1 (sVEGFR1) and sVEGFR2 in 133 patients with acute myeloid leukemia (AML) and in 80 patients with myelodysplastic syndromes (MDS) at the time of initial presentation and compared clinical behaviors.

Results: A reverse correlation was observed between plasma sVEGFR1 levels and the rate of complete remission (CR) in patients with AML, but not in patients with MDS.

Relative increase in leukemia-specific DNA in peripheral blood plasma from patients with acute myeloid leukemia and myelodysplasia.

Using loss of heterozygosity (LOH) and X-chromosome inactivation, we compared peripheral blood (PB) plasma with bone marrow (BM) cells in detecting genomic abnormalities in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We detected LOH in the PB plasma of all 45 patients who had cytogenetically documented chromosomal abnormalities (5q-, 7-, +8, 17-, or 20-). BM cells from the same patients showed LOH in 89% of patients with MDS and 70% of patients with AML.

Use of plasma DNA in detection of loss of heterozygosity in patients with multiple myeloma.

Background: Deletions or structural abnormalities in chromosomes 11 and 13 have been shown to be important in predicting clinical behavior in patients with multiple myeloma (MM). However, cytogenetic analysis in MM is frequently difficult because of poor yield of informative metaphases and the disease is frequently patchy, which complicates fluorescent in situ hybridization studies.

Objectives: The purpose of this study was to explore the potential of using peripheral plasma DNA for the detection of loss of heterozygosity (LOH) in chromosomes 11 and 13 in patients with MM.

Transient down-modulation of CD20 by rituximab in patients with chronic lymphocytic leukemia.

Lymphoid cells in most patients with chronic lymphocytic leukemia (CLL), when treated with rituximab, become CD20-. This is thought to be due to masking of CD20 by rituximab. We used specific antimouse immunoglobulin antibodies to detect rituximab on the surface of CLL lymphocytes and we demonstrate that rituximab is rarely detectable after therapy.

In vitro activity of dimethylarsinic acid against human leukemia and multiple myeloma cell lines.

Purpose: Arsenic trioxide (As(2)O(3)), an inorganic arsenic compound, has recently been approved for the treatment of relapsed or refractory acute promyelocytic leukemia. However, systemic toxicity associated with As(2)O(3) treatment remains a problem. Inorganic arsenic is detoxified in vivo by methylation reactions into organic arsenic compounds that are less toxic.