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Keywords:

  • Tie-1;
  • chronic myeloid leukemia;
  • expression;
  • prognostic factor;
  • angiogenesis;
  • bone marrow

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

The Tie-1 tyrosine kinase receptor and its thus far unidentified ligand appear to play a distinct role in the regulatory pathways of early hematopoiesis and angiogenesis. Because vascularity is increased in the bone marrow of patients with chronic myeloid leukemia (CML), the authors evaluated the clinical significance of Tie-1 expression in such patients.

METHODS

Using Western blot analysis and solid-phase radioimmunoassay (RIA), the authors quantified Tie-1 protein in bone marrow samples from 128 patients with CML and 31 normal controls.

RESULTS

The median Tie-1 RIA value of CML samples was significantly higher than in normal controls (P = 0.01). The authors found no significant differences in Tie-1 levels in patients with early chronic, late chronic, accelerated, and blastic phases (P = 0.2). High Tie-1 levels correlated with short survival in patients with early chronic phase (P = 0.003; Cox proportional hazard model) but not in patients with late chronic (P = 0.2) or accelerated/blastic (P = 0.2) phase CML. Tie-1 protein level was also prognostic when patients were separated into two groups by the median value. High Tie-1 level in early chronic phase was associated with significantly shorter survival than low Tie-1 level (median survival, 116 vs. 61 months; P = 0.03). In patients with early chronic phase CML, Tie-1 levels correlated directly with patient age (P = 0.004) and platelet count (P = 0.003), and inversely with leukocyte count (P = 0.007). Tie-1 as a predictor of survival in early chronic phase CML was independent of risk group, spleen size, age, hemoglobin, and basophil count (P = 0.03; multivariate Cox proportional hazard model).

CONCLUSIONS

The authors' findings support the hypothesis that angiogenesis may play a major role in the pathophysiology of chronic phase CML. Cancer 2002;94:1517–21. © 2002 American Cancer Society.

DOI 10.1002/cncr.10363

Increased bone marrow vascularity recently has been reported in patients with several hematologic malignancies, including acute myeloid leukemia (AML), myelodysplastic syndrome, and myeloproliferative diseases (e.g., chronic myeloid leukemia [CML]).1–6 Many molecules have been isolated that regulate the processes of vasculoangiogenesis. Among them, two ligand/receptor systems are characterized by their largely endothelial specific expression. One system includes vascular endothelial growth factor (VEGF) and VEGF receptor 1, 2, and 3.7 The other includes Tie-1 receptor, with its yet unidentified ligand, and Tie-2 receptor, with its ligands angiopoietin 1 and 2.8

In hematologic malignancies, the expression and significance of VEGF and its receptors have been documented.9–11 In CML, the number of VEGF positive bone marrow cells was significantly higher than in normal controls and correlated with bone marrow vascularity.4 VEGF protein expression in CML was also significantly increased.1

The role and/or clinical significance of other factors or receptors, including Tie-1, Tie-2, and angiopoietins, possibly involved in the vascularization and pathophysiology of hematologic malignancies is not elucidated. Tie-1 is a tyrosine kinase receptor expressed in all vascular endothelial cells.8 It also is expressed in umbilical cord blood, hematopoietic bone marrow, and stem cell populations as well as some bone marrow B cells.12, 13 Its expression is up-regulated during in vitro hematopoietic stem cell megakaryocytic differentiation.13 Tie-1 initially was found to be expressed in megakaryoblastic leukemia cell lines,14 but more recent studies found Tie-1 expression in leukemic cells derived from various myeloid cell lineages.15, 16 The role, if any, that Tie-1 plays in the pathophysiology or propagation of myeloid cell diseases is unknown. In this study, we investigated the expression of Tie-1 protein in bone marrow samples obtained from patients with CML and analyzed its clinical and prognostic significance.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients and Controls

Pretreatment cellular Tie-1 protein concentrations were measured in bone marrow samples from 128 patients with CML at the time of presentation to the University of Texas M. D. Anderson Cancer Center. We also measured Tie-1 protein concentrations in 31 normal bone marrow samples (obtained as a part of staging from patients with suspected lymphoma but without evidence of lymphoma in the bone marrow). All samples were obtained under protocols approved by the Internal Review Board and with the patients' written informed consent. The characteristics of patients are shown in Table 1. All patient data were collected by reviewing patients' records and from the Department of Leukemia database.

Table 1. Clinical Characteristics of Patients with CML
Disease phaseNo. of patients
  1. CML: chronic myeloid leukemia.

Early chronic76
Late chronic23
Accelerated21
Blastic8
Characteristics of patients with early chronic phase CML%
Age ≥ 60 ys10
Spleen ≥ 10 cm below costal margin5
Leukocyte count ≥ 30 × 109/L33
Platelet count ≥ 700 × 109/L12
Peripheral blasts ≥ 3%2
Bone marrow blasts ≥ 5%1
Peripheral basophils ≥ 7%7
Bone marrow basophils ≥ 3%15

Protein Extraction

Cell pellets were lysed on ice for 30 minutes in TENN buffer (50 mmol/L Tris-HCL [pH 7.4], 5 mmol/L ethylenediamine tetraacetic acid, 0.5% Nonidet P-40, and 150 mmol/L NaCl supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, 2 μg/mL leupeptin, and 2 μg/mL pepstatin). After vortexing, samples were left on ice for 1 hour. Lysates were clarified by microcentrifugation for 1 hour at 14,000 rpm. The protein concentration was determined by the Bradford method. Two hundred micrograms of cell extract was subjected to 9.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue R-250 to check the protein profile and amount of protein loaded.

Western Blot Analysis

Tie-1 protein was analyzed using the Western blotting technique. For each sample, 200 μg of cell extract was electrophoretically separated on a 9.5% SDS-PAGE gel and transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked for 4–6 hours at room temperature with 5% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 and 0.01% sodium azide. The blots then were incubated overnight at 4 °C with rabbit anti-Tie1 antibody (Santa Cruz Biotech, Santa Cruz, CA) at a concentration of 1 μg/mL in PBS containing 2.5% nonfat milk, 2.5% bovine serum albumin (BSA), and 0.1% Tween-20. The membranes were washed with PBS containing 0.1% Tween-20 and incubated with anti-rabbit immunoglobulin (Ig) linked to horseradish peroxidase (Sigma Chemical Co., St Louis, MO) diluted 1:2000 in PBS containing 1% nonfat milk and 0.1% Tween-20. Immunoreactive bands were developed using the ECL detection system (Amersham, Arlington Heights, IL). The membranes then were stripped of the primary and secondary antibodies under conditions recommended by the manufacturer and blocked and probed with antiactin IgM monoclonal antibody (Amersham) to check for equal loading of protein in each lane.

Solid-Phase Radioimmunoassay

Solid-phase radioimmunoassay (RIA) was used to measure Tie-1 protein. Microtiter plates were coated with 5 μg of protein, extracted from patients' samples, in 50 μL of PBS and left overnight at 4 °C. The plates then were washed with PBS and blocked with 100 μL of 1% BSA in PBS for 1 hour at 37 °C. The plates were incubated overnight at 4 °C with 50 μL of rabbit anti-Tie1 antibody diluted 1:1000 in PBS containing 0.1% BSA. The plates then were washed with PBS and amplified with goat anti-rabbit IgG antiserum (Sigma Chemical Co.) and diluted 1:1000 in 0.1% BSA in PBS for 2 hours at 37 °C. After washing, the plates were developed for 2 hours at room temperature using excess 125I-labeled protein G (200,000 cpm [50 IU] in 0.1% BSA and PBS per well). The plates then were washed with PBS, and the samples were separated into individual wells. Radioactivity in each well was measured with a gamma counter (LKB Biotechnology, Uppsala, Sweden). The assays were performed in triplicate, and the results were corrected for the nonspecific binding detected in control wells (1–2%), which were not coated with a test antigen but were blocked with BSA. A second set of plates was incubated with antiactin antibodies to confirm the use of an equal amount of total cellular protein from each sample. Each sample was tested in triplicate, and this was repeated twice in two different experiments. No significant differences were observed between measurements. Normal samples were included with each experiment, and each plate contained two or more normal samples.

Statistical Considerations

Associations among variables were assessed using the Spearman rank correlation analysis. The Kruskal–Wallis test was used to compare various groups of data. Survivals were plotted when cutoff points were used by the Kaplan–Meier method and compared using the log-rank test. Cox proportional hazard model was used to study correlations with survival when Tie-1 level was used as a continuous variable.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Assessment of Cellular Tie-1 Protein Levels in Bone Marrow of Patients with CML

We found a detectable level of Tie-1 protein in all the bone marrow samples examined using RIA. However, Western blot analysis did not show detectable Tie-1 protein in all samples (Fig. 1). Radioimmunoassay values indicating levels of Tie-1 protein in bone marrow samples from 128 patients with CML were normalized to the median Tie-1 level in 31 normal bone marrow samples, which was assigned a value of 1. The median Tie-1 RIA value the of 128 CML samples was 1.3, significantly higher than in normal controls (P = 0.001). We found no significant differences between Tie-1 levels in bone marrow of 76 early chronic, 23 late chronic, 21 accelerated, and 8 blastic phase CML patients (P = 0.2).

thumbnail image

Figure 1. Tie-1 protein expression in bone marrow samples from patients with CML. Representative Western blot showing Tie-1 protein expression in samples from patients with CML. Relative values of individual samples obtained by solid-phase RIA are indicated. CML: chronic myeloid leukemia; RIA: radioimmunoassay.

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Clinical Significance of Tie-1 Protein Expression

Using Cox proportional hazard model for correlation analysis and Tie-1 protein level as a continuous variable, we found Tie-1 levels to correlate inversely with the length of survival of patients in early chronic (P = 0.003), but not in late chronic (P = 0.2) or accelerated/blastic (P = 0.2) phases. We then assessed this relation using the median value of Tie-1 as a cut-off point. The median Tie-1 value in early chronic phase CML was 1.2. Patients in early chronic phase with high Tie-1 protein levels had a significantly shorter survival than those with low Tie-1 levels (P = 0.03; Fig. 2). Correlation analyses of Tie-1 levels and patient characteristics (Table 2) showed an inverse correlation between Tie-1 levels and leukocyte counts (P = 0.007; r = −0.3) and a direct correlation with platelet counts (P = 0.003) and patient age (P = 0.004). Tie-1 level did not correlate with the percentage of peripheral or bone marrow blasts (P = 0.6 and P = 0.3, respectively).

thumbnail image

Figure 2. Survival of patients with early chronic phase chronic myeloid leukemia (CML) based on pretreatment bone marrow Tie-1 protein level. Tie-1 protein was measured in bone marrow samples from 76 patients with early chronic phase CML. Survival time of patients is presented in relation to Tie-1 level.

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Table 2. Correlation between Tie-1 Expression and Characteristics of Patients with Early Chronic Phase CML
Patient characteristicP value
  • CML: chronic myeloid leukemia.

  • a

    r = −0.3.

  • b

    r = 0.3.

  • c

    r = 0.3.

Age0.004a
Hemoglobin level0.9
Leukocyte count0.007b
Platelet count0.003c
Peripheral blast percentage0.6
Peripheral basophil percentage0.1
Bone marrow blast percentage0.3
Bone marrow basophil percentage0.8

Univariate and multivariate analysis assessing correlations between standard prognostic variables and Tie-1 level in patients with early chronic phase CML and their survival is shown in Table 3. Tie-1 as a predictor of survival in early chronic phase CML was independent of risk group, spleen size, age, hemoglobin, and basophil count (P = 0.03; multivariate Cox proportional hazard model).

Table 3. Correlation between Characteristics of Patients in Early Chronic Phase CML and Survival
CharacteristicaUnivariate analysis (P value)Multivariate analysis (P value)
  • CML: chronic myeloid leukemia.

  • a

    Studied as continuous variables (except “risk group” that was studied as a categoric value).

Age0.050.3
Spleen size0.070.5
Leukocyte count0.20.2
Platelet count0.050.6
Hemoglobin level0.90.2
Peripheral basophil percentage0.40.7
Risk group0.40.8
Tie-10.0030.03

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Our report is the first study to our knowledge of the prognostic significance of Tie-1 receptor expression in CML. Two previous studies evaluated Tie-1 receptor expression in CML. Muroi et al.17 analyzed Tie-1 expression by flow cytometry in peripheral blood mononuclear cells (>80% leukemic cells) obtained from 4 patients with blastic phase CML and found no positive samples. Kukk et al.18 reported Tie-1 expression in 6 of 11 CML blastic phase peripheral blood samples; expression was low in all cases. Although the presence of Tie-1 receptor was documented previously in peripheral blood leukemic cells from some patients with CML, its expression in the bone marrow of patients with CML could be the function of increased bone marrow vascularity.1, 4 It recently has been suggested that CML arises from a hemangioblastic progenitor cell, the progeny of which are malignant blood cells and genotypically clonal endothelial cells.19 If correct, then malignant endothelial cells might play a major role in the reported increased vascularity in the bone marrow of CML patients. Furthermore, the level of expression of endothelial cell surface marker may be reflected in the disease activity.

Our analysis of Tie-1 protein expression in bone marrow samples from a large group of patients using a sensitive technique showed detectable levels of Tie-1 protein in all samples examined. We found Tie-1 levels not to be differentially expressed in different phases of CML, that is, not to be associated with disease progression. The expression of Tie-1 in patient samples was significantly higher than in normal samples. Among patients with early chronic phase CML, high Tie-1 expression was independently associated with shorter survival. Certainly, the role of the Tie-1 ligand-receptor system in the leukemogenic process or the biology of CML needs further investigation, but the prognostic significance of abnormally high Tie-1 expression in early chronic phase CML patients is instructive as the new knowledge regarding the pathophysiology of CML, the possible involvement of angiogenic factors and receptors, like Tie-1, may help developing new angiogenesis related therapeutic strategies.

Immunohistochemistry may provide important information on the cell type that express the Tie-1 protein and comparison of the role of Tie-1 on leukemic cells with its role on endothelial cells may help in understanding the complex role of angiogenic factors in leukemias.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES