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. MATERIALS AND METHODS: Peripheral blood plasma samples from 41 patients with confirmed acute myeloid leukemia with intermediate or poor cytogenetics were obtained prior to induction therapy for proteomic analysis. For each plasma sample, four fractions eluted from a strong anion column were applied to 3 different ProteinChip array surfaces and 12 surface-enhanced laser desorption/ionization spectra were generated. Peaks that correlated with response were identified, and decision trees incorporating these peaks along with various clinical and laboratory findings were constructed to predict response. RESULTS: Multiple decision trees were constructed. One peak, when combined with age, provided strong positive prediction of responders with 83% accuracy. A second tree, which combined one peak with both cytogenetics and the percent of monocytes in peripheral blood, detected responders with 95% accuracy. A third peak was adequate to predict responders in the intermediate cytogenetic group with 86% accuracy. CONCLUSIONS: Proteomic analysis should be further explored to define factors important in predicting clinical response in patients with acute myeloid leukemia.

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. Plasma testing allowed the detection of a novel 4 bp deletion in NPM1 in one patient. Analysis of clinical data corroborated previous data linking NPM1 mutations with better clinical outcome. These data underline the significance of NPM1 in the biology and clinical behavior of AML, and demonstrate the reliability and efficacy of plasma-based testing for NPM1 mutations.

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). RESULTS: All activity levels were significantly higher in patients who had CLL compared with the levels in a control group of healthy volunteers (P<.001). Rai stage was correlated with Ch-L activity (P<.001) but not with Cas-L or Tr-L activity. Levels of beta2 microglobulin (B2M) were correlated with Ch-L activity (correlation coefficient [R]=0.4; P<.001) and with Cas-L activity (R=0.25; P=.001) but not with Tr-L activity. Cas-L activity as a continuous variable was a strong predictor of survival. Ch-L and Cas-L activity levels as categorical variables both were strong predictors of survival; Cas-L activity was independent of B2M level but not of immunoglobulin variable heavy chain gene (IgVH) mutation status. However, the combination of elevated B2M levels (>3.2 mg/L) and Cas-L activity (>1.32 pmoL/second/mL plasma) was associated with significantly shorter survival independent of IgVH mutation status. CONCLUSIONS: The current results indicated that measuring plasma proteasome activity has prognostic value in CLL that, when combined with B2M, can be independent of IgVH mutation status.

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. Resistant patients had significantly lower levels of BCR-ABL, CrkL and AKT phosphorylation than previously untreated patients, but STAT5 phosphorylation showed no difference. These observations suggest that imatinib- resistance is not necessarily dependent on higher activity in BCR-ABL-dependent pathways, but is likely due to the activation of other pathways.

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. This method also allows the measurement of the level of phosphorylation of the immunoprecipitated BCR-ABL using antibodies against phosphorylated ABL protein, which can be used for monitoring of therapy with kinase inhibitors. The sensitivity of this immunoassay was comparable to the sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) assay. This technique is useful in monitoring patients with chronic myeloid leukemia (CML) or acute lymphoblastic leukemia (ALL), but the same approach can be used in other translocations and has the potential of multiplexing.

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. 17-AAG reduced HSP90 levels, but increased HSP70 levels. Phospho-STAT5, total STAT5, and total AKT levels were also reduced by 17-AAG treatment. Further, phospho-JAK2, total JAK2, and cell viability were reduced to a greater extent by 17-AAG than by the pan-JAK kinase family inhibitor JKII or the JAK2-specific inhibitor AG490, and these inhibitors failed to synergize with 17-AAG. Flow-cytometry-based assays for JAK/STAT signaling pathway and HSPs are likely to have broad clinical utility for monitoring patients with abnormalities in the JAK2 pathway.

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. Samples from 160 CML patients and 180 control individuals without CML were tested. Cells and plasma samples from 93 of the CML patients were re-analyzed 3-12 months after imatinib treatment. RESULTS: Ratios of BCR-ABL:ABL mRNA in paired cell and plasma samples of the 160 CML patients correlated significantly (r=0.83; p<0.001). When results were compared directly using the sign test, the pre-therapy plasma results were significantly different from those from peripheral blood cells (p=0.028), but not bone marrow cells (p=0.119). Absolute levels of BCR-ABL mRNA in plasma strongly correlated with many laboratory characteristics in pre-therapy CML patients. Higher BCR-ABL: ABL ratios were detected in plasma samples at all time points after treatment, although this was significant only at 3 months (p=0.0003). In cases in which results from the assays disagreed, minimal residual disease was detected in plasma samples significantly more frequently than in cell samples (p<0.001). INTERPRETATION AND CONCLUSIONS: Plasma was a reliable source for monitoring BCR-ABL mRNA levels. Minimal residual disease detection from plasma was more sensitive than from cell samples. Our results suggest that absolute levels of BCR-ABL mRNA per unit volume of plasma may reflect tumor load.

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. The expression of specific marker or confirmation of markers defines a specific cell population or the original of these cells. This in turn helps in diagnosis and classification of various hematological diseases and leads to choosing a specific therapy. Here, we describe a methodology for using flow cytometry with six colors for the analysis of various tissues for hematological diseases.

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. We describe a quantitative flow cytometry approach to measuring levels of the CD20 surface antigen, the molecular target of rituximab.

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. We describe a flow cytometry method for quantitation of total and phosphorylated intracellular proteins. Compared with Western blot analysis, this technique dramatically decreases time and labor while providing multiparameter information on specific cell populations. As an example, total and phosphorylated CRKL is quantitated. The methodology has the potential for widespread application in the monitoring of targeted therapy.

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. We describe a cell-free, bead-based method that takes advantage of this phenomenon. Our approach is based on immunoprecipitation of the protein of interest on the surface of beads, followed by detection of the protein or its modification (phosphorylation) using a secondary antibody labeled with phycoerythrin at a 1:1 ratio. Fms-like tyrosine kinase-3, which is mutated in majority of cases of acute myeloid leukemia, is used as an example. This method could be applied to the quantitation of several other proteins without the need for intact cells.

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. We describe a novel bead-based assay that measures circulating alemtuzumab by taking advantage of remnant rat sequence in the antibody. Levels of total alemtuzumab complexed with CD52, and free alemtuzumab are quantitated in the serum or plasma by flow cytometry. This approach is applicable to the measurement of other humanized antibodies that contain an appropriate remnant animal sequence.

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. Here, we describe a novel immunoassay that directly measures levels of BCR-ABL fusion protein and its phosphorylation in peripheral blood plasma and cell lysates. This approach has the potential for widespread application in the detection and quantitation of other fusion genes involved in hematological malignancies.

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. NHL and HD patients had significantly higher levels of sHLA-1 and beta2M than control subjects. In NHL patients, sHLA-I levels correlated with clinical behavior in a fashion similar to that of beta2M. However, multivariate analysis incorporating beta2M, sHLA-I, and international prognostic index (IPI) indicated that NHL patients with elevated (>312.6mug/100mL) sHLA-I levels had significantly shorter survival, independent of IPI score as well as beta2M. In HD patients, beta2M but not sHLA-I levels were associated with clinical behavior. These findings not only establish the role of sHLA-I as an independent tumor marker in NHL that can be used to stratify patients, but also suggest that beta2M and sHLA-I may reflect different biological processes in HD and NHL. Further studies are needed to assess whether the immunomodulatory properties of sHLA-I may be responsible for its divergence from beta2M as an indicator of clinical behavior in HD.

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. Follow-up continued for a median period of 71 weeks. For each plasma sample, 4 fractions eluted from a strong anion column were applied to 3 different ProteinChip array surfaces, and 12 surface-enhanced laser desorption/ionization (SELDI) spectra were generated. Peaks that correlated with recurrence were identified and decision trees were constructed and evaluated, using only 2 peaks per predictive tree. RESULTS: The best decision trees provided strong positive prediction of recurrence, with correct predictions 84% to 92% of the time, whereas negative prediction of patients who did not experience recurrence was less robust, with 62% to 74% accuracy. Prediction of recurrence was independent of cytogenetics, bone marrow blast count, lactate dehydrogenase, beta-2-microglobulin, or surface markers. Positive prediction of L3 morphological classification was achieved in 80% of test cases. CONCLUSIONS: Peripheral blood plasma is adequate to predict clinical behavior in ALL patients irrespective of the percentage of bone marrow blasts. Proteomic analysis of plasma offers a useful approach for profiling patients with ALL.

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 < .001), and the absence of mutation in the immunoglobulin heavy chain variable region (IgV(H)) (P < .001), and were inversely correlated with platelet count (P = .002). We found that TPO correlated strongly in a continuous manner with overall survival in both previously treated and untreated patients. The univariate Cox proportional hazard model demonstrated that high TPO levels were associated with shorter survival (P < .001), and multiple variable Cox proportional hazards regression analysis demonstrated that this was independent of the IgV(H) mutation status, beta2-M, and Rai stage. Recursive partitioning showed that a cutoff point of 639 pg/mL separated the CLL patients into 2 major survival groups (P < .001). The effects of beta2-M were masked by the effects of TPO in the patients with TPO levels higher than 639 pg/mL, but in the remainder, patients with beta2-M level higher than 4.95 mg/L had significantly shorter survival than those with lower values. Plasma TPO and beta2-M may be useful for the prediction of clinical behavior in CLL and may replace the need for the determination of IgV(H) mutation status.

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. Plasma levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-1 receptor alpha (IL-1Ralpha), IL-6, IL-8, VEGF receptors VEGFR1 and VEGFR2, and thrombopoietin (TPO) were measured in plasma samples of 95 patients by enzyme-linked immunosorbent assay (ELISA). In a univariate Cox proportional hazards model, higher levels of IL-1Ralpha, IL-8, VEGFR1, and VEGFR2 were predictive of poor survival. In contrast, higher levels of VEGF were predictive of longer survival, and higher levels of bFGF suggested a similar trend (P = .09). The multivariate model simultaneously included VEGF (relative risk [RR] for death, 8.01; P = .001 for levels less than or equal to 19.5 pg/mL), IL-1Ralpha (RR, 5.12; P = .007 for levels greater than 373 pg/mL), and VEGFR2 (RR, 4.01; P = .04 for levels greater than 8222 pg/mL) as independent factors for survival. Of interest is the association of high levels of VEGF with good prognosis and higher levels of VEGF receptors with poor outcome. These data reflect the complexity by which angiogenic factors may affect the clinical behavior of patients with ALL, and this complexity should be considered in any therapeutic strategy incorporating antiangiogenic agents.

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. We developed a simple sandwich enzyme-linked immunosorbent assay (ELISA) to measure alemtuzumab that takes advantage of the remaining rat sequence in alemtuzumab. Using specific anti-rat immunoglobulin (Ig) antibodies (absorbed against human Ig), alemtuzumab levels were measured in the serum and plasma of patients treated with alemtuzumab. Levels were similar between plasma and serum samples, in fresh samples and samples stored at 4 degrees C for 24 h, but were significantly lower in samples stored at room temperature for 24h. The assay was successfully used to determine serum alemtuzumab pre- and post-treatment. This assay is simple and adaptable for high throughput testing, with a limit of detection of 0.05 microg/ml and a coefficient of variation of +/-12.5%. No false positivity was observed in >200 samples tested. This validated assay should help optimize the dosing and scheduling of alemtuzumab therapy. The underlying principles are also applicable to the measurement of other humanized antibodies using an appropriate anti-Ig.

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. METHODS: The authors used Western blot analysis, immunoprecipitation, and enzyme-linked immunoadsorbent assay to investigate the presence of soluble CD52 (sCD52) in the plasma specimens of 117 patients with CLL. They also used in vitro mixing experiments to examine the ability of sCD52 to compete with cells and sequester therapeutic alemtuzumab. RESULTS: The authors detected high levels of sCD52 in the plasma specimens of patients with CLL. sCD52 can compete with cells in vitro for binding to alemtuzumab, and can form complexes in patients receiving alemtuzumab. Plasma levels of sCD52 were found to be correlated (r)with Rai stage (P = 0.0001), beta-2-microglobulin (beta-2M) levels (P = 0.00002), soluble CD23 levels (r = 0.42, P < 0.001), and immunoglobulin mutation status (P = 0.003). In the multivariate analysis adjusted for beta-2M level, patients with sCD52 levels > 2336 nM/L had a nearly 4-fold increase in risk of death. Higher levels of plasma alemtuzumab were achieved when levels of sCD52 were lower. CONCLUSIONS: These data not only demonstrated that sCD52 was detectable and useful in the staging and monitoring of patients with CLL, but also showed that sCD52 formed immune complexes with alemtuzumab and may influence the efficacy and toxicity of alemtuzumab therapy.