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Increased angiogenesis and Fas-ligand expression are independent processes in acute myeloid leukemia
Leukemia Research 25 (2001) 1067– 1073 www.elsevier.com/locate/leukres
Increased angiogenesis and Fas-ligand expression are independent processes in acute myeloid leukemia Je-Jung Lee a, Ik-Joo Chung a, Moo-Rim Park a, Dong-Wook Ryang b, Chang-Soo Park c, Hyeoung-Joon Kim a,* a b
Department of Internal Medicine, Chonnam National Uni6ersity Medical School, 8 Hak-dong, Dong-ku, Kwangju 501 -757, South Korea Department of Clinical Pathology, Chonnam National Uni6ersity Medical School, 8 Hak-dong, Dong-ku, Kwangju 501 -757, South Korea c Department of Pathology, Chonnam National Uni6ersity Medical School, 8 Hak-dong, Dong-ku, Kwangju 501 -757, South Korea Received 10 May 2000; accepted 30 April 2001
Abstract We evaluated the clinical significance of tumor angiogenesis and Fas-ligand (FasL) expression using parameters including the microvessel count (MVC), vascular endothelial growth factor (VEGF) level, and FasL expression in patients with acute myeloid leukemia (AML). Paraffin-embedded bone marrow (BM) sections from 43 AML patients at diagnosis, 20 patients after subsequent induction therapy, and 18 controls with non-invasive lymphoma were stained immunohistochemically for von Willebrand factor (vWF) and FasL. VEGF in BM mononuclear cells from 32 AML patients at diagnosis and 10 controls, including bone marrow transplantation donors, was assayed by an ELISA method. We found that the mean MVC, VEGF level, and FasL expression in AML patients at diagnosis were significantly higher than those of controls, with a significant correlation between the MVC and VEGF levels (r= 0.43). However, there were no correlations between FasL expression and MVC or VEGF level. The mean MVC and FasL expression after induction therapy were lower than those evaluated at diagnosis, but were higher than those of controls. There was a correlation between the MVC and percentage of BM blasts (r= 0.479), but no correlation between the MVC, VEGF level, or FasL expression and other hematologic or clinical variables. Our findings provide evidence of increased angiogenesis and tumor immune escape in AML, and both angiogenesis and tumor immune escape are independent processes in AML. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Microvessel count; VEGF; Tumor immune escape; Fas-ligand; AML
1. Introduction Angiogenesis plays an important role in the growth of primary tumors and metastatic sites of solid tumors, and is an independent clinical prognostic factor in Abbre6iations: AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ara-C, cytarabine; ATRA, all-trans retinoic acid; BM, bone marrow; BMT, bone marrow transplantation; CML, chronic myeloid leukemia; CR, complete remission; DLI, donor lymphocyte infusion; FAB, French–American–British; FasL, Fas-ligand; IDA, idarubicin; MDS, myelodysplastic syndrome; NK, natural killer; MNC, mononuclear cell; MVC, microvessel count; MVD, microvessel density; SD, standard deviation; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor. * Corresponding author. Tel.: +82-62-2206-572; fax: + 82-622258-578. E-mail address: [email protected] (H.-J. Kim).
several malignancies [1–3]. A balance between angiogenic stimulators and angiogenesis inhibitors tightly regulates angiogenesis in endothelial cells [1,4]. Of the proteins known to activate endothelial growth and movement, vascular endothelial growth factor (VEGF) seem to be one of the most potent mitogens for endothelial cells in the neovascularization of solid tumors and is associated with enhanced angiogenesis, metastasis, and poor outcome in solid tumors [1,2]. Recently, several investigators evaluated angiogenesis and VEGF in hematologic malignancies, such as multiple myeloma, myelodysplastic syndrome (MDS), leukemia, and lymphoma, but its exact role in pathogenesis is still unclear [5–10]. Fas-ligand (FasL) is a 37-kDa transmembrane protein that induces apoptosis by killing Fas-positive target cells through both juxtacrine and paracrine
0145-2126/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(01)00082-0
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mechanisms [11,12]. Recently, FasL expression was found in several types of solid tumors or tumor cell lines [13]. Abnormal FasL expression in these tumor cells induces the killing of activated tumor-specific Tcells or natural killer (NK) cells bearing Fas at the tumor site, resulting in escape from tumor immune surveillance [13,14]. Recently, some investigators have reported that leukemic cells also have a mechanism of tumor immune escape involving the Fas/FasL pathway [12,15– 17]. The relationship and balance between angiogenesis and apoptosis in growth of solid tumors is still unclear. Hypoxia, which is a potent stimulator of angiogenesis, is closely correlated with factors known to induce apoptosis [18,19]. Based on this finding, some investigators reported an inverse correlation between angiogenic factors and apoptotic factors in gastric and lung cancer [20,21], but any correlation was denied in breast cancer [22]. In hematopoietic cells, Katoh et al. [23,24] reported that VEGF inhibits apoptotic cell death by producing ZK7 protein in vitro. However, direct comparison between angiogeneic factors and FasL expression, one of the mechanisms for tumor immune escape, has not been extensively studied in acute myeloid leukemia (AML). In this study, we evaluated the clinical significance of angiogenesis, VEGF, and FasL expression in patients with AML.
2. Materials and methods
idarubicin (IDA) (12 mg/m2/day IV over 30 minutes on days 1–3) and cytarabine (ara-C) (100 mg/m2/day IV continuously on days 1–7) for non-APL and all-trans retinoic acid (ATRA) (45 mg/m2 orally until complete remission) and/or IDA (12 mg/m2/day IV over 30 min on days 2, 4, 6, and 8) for APL.
2.2. Immunohistochemistry We used the avidin-biotin-peroxidase complex method of immunohistochemical staining with a Microprobe Immuno/DNA stainer (Fisher Scientific, Pittsburgh, PA). Three-mm-thick BM sections were mounted on slides and deparaffinized. For FasL staining, the sections were heated in a microwave oven in 2.1% citric acid buffer solution (pH 6.0) for 7 min to retrieve the antigens. For vWF staining, sections were incubated with Pepsin solution (Research Genetics, Huntsville, AL) for 3 min at 45 °C. After preincubation with 0.6% hydrogen peroxide, protein blocker, and anti-biotin for 5 min at 45 °C, the sections were incubated in the primary antibody for vWF (1:500 working concentration) for 15 min at 45 °C or FasL (1:100 working concentration) for 90 min at room temperature in a moist chamber. After washing, the sections were incubated in biotinylated anti-mouse IgG (Sigma, St Louis, MO) for 7 min at 45 °C. The streptavidin-horseradish peroxide detection system (Research Genetics) was applied for 7 min at 45 °C. The sections were developed with the chromogen 3-amino-9-ethylcarbazole (AEC) for 7 min at 45 °C. Counter staining was performed with hematoxylin for 1 min at 45 °C.
2.1. Patients 2.3. Determination of micro6essel count (MVC) Paraffin-embedded bone marrow (BM) sections from 43 patients with newly diagnosed AML and 20 patients who received remission induction therapy at Chonnam National University Hospital from May 1999 to January 2000 were stained immunohistochemically for von Willebrand factor (vWF) (Dako, Carpinteria, CA) and FasL (Santa Cruz Biotechnology, Santa Cruz, CA). Control specimens were left over biopsed samples from 18 patients with lymphoma for staging with normal BM morphology. In addition, BM mononuclear cells (MNCs) from 32 patients with newly diagnosed AML were assayed for VEGF by an ELISA method (R&D Systems, Minneapolis, MN). The controls for VEGF were MNCs from 10 healthy donors for allogeneic bone marrow transplantation (BMT). The median age of the patients was 45 years (range 16–78). There were 24 males and 19 females. According to the French–American – British (FAB) classification, 10 patients had acute promyelocytic leukemia (APL) and 33 patients had non-APL. Of 43 patients, 18 patients with non-APL and eight patients with APL received remission induction therapy, which consisted of
After selecting the two areas with the highest microvessel density (MVD) on a ×200 field, the MVC was assessed in ×400 fields. Areas of staining with no discrete breaks were counted as a single vessel. Large vessels (] 10 mL) or vessels in the periosteum or bone were excluded.
2.4. VEGF ELISA BM MNCs were separated by centrifugation using Ficoll-Hypague (Lymphoprep™, Nycomed, Oslo, Norway). The cells were sonicated with three 15-s, 60 W-bursts of a Fisher sonic dismembrator, and subsequently clarified by centrifugation at 12,000 rpm for 10 min at 4 °C. For each sample, 50 mg of protein measured with the BCA protein assay kit were tested using sandwich ELISA according to the manufacturer’s instruction (R&D Systems, USA), and all analyses and calibrations were carried out in duplicate. Samples (200 mL) and standards (200 mL) were added to a coated microtiter plate and incubated for 2 h at room temper-
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ature. After washing with buffer solution three times, 200 mL of recombinant anti-VEGF polyclonal antibody conjugated to horseradish peroxide were added to the wells. After an additional 2-h incubation, the wells were rinsed again, and 200 mL of hydrogen peroxide and tetramethylbenzidine were added. The reaction was stopped by the addition of 50 mL of 2 N sulfuric acid. The absorbance of each well was measured using a microplate reader at 450 nm and plotted against a standard curve with VEGF levels expressed in pg/mL.
2.5. Statistical analysis All statistical analyses were performed with the program SPSS 10.0 (SPSS, Chicago, IL). The MVC, VEGF level, and FasL expression of the patients and controls were compared using the Mann-Whitney U-test or the Kruskal-Wallis test. Correlations between clinical or hematological variables and MVC, VEGF level, or FasL expression used Pearson’s test for parametric variables and Spearman’s z test for nonparametric variables. P-valuesB 0.05 were considered statistically significant. The results are expressed as the mean plus or minus the standard deviation (SD).
3. Results
3.1. MVC, VEGF le6el, and FasL expression are increased in AML Using vWF staining (Fig. 1), the mean MVC was 31.99 14.3 vessels/ × 400 field (range 9-71) in newly diagnosed AML patients, 18.697.9 vessels/ × 400 field (range 6–35) in patients after induction therapy, and 9.1 9 2.8 vessels/ × 400 field (range 4– 14) in controls. The MVC in patients with newly diagnosed AML was significantly higher than in controls (PB 0.001). The mean MVC in patients after induction therapy was significantly lower than that of newly diagnosed AML patients (PB 0.001), but was still higher than that of controls (PB0.001). However, patients with newly diagnosed AML showed wide ranges of the MVC. In addition, the cytoplasm of megakaryocytes stained strongly for vWF (Fig. 1). With FasL staining (Fig. 2), the mean expression of FasL in newly diagnosed AML patients, patients after induction therapy, and controls was 55.19 27.2 (range 5–95), 39.3924.4 (range 10– 85), and 12.89 10.3% (range 3–35), respectively. The differences between the expression of FasL in newly diagnosed AML patients and controls were highly significant (P B 0.001). The mean expression of FasL in patients after induction therapy was lower than that of newly diagnosed AML patients (P= 0.037). Although the mean expression of FasL in patients after induction therapy was still higher
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than that of controls (PB 0.001), many patients underwent induction therapy showed decreased FasL expression. However, there were wide ranges of FasL expression within each group. The FasL staining was positive in lymphocytes, myeloblasts, erythroblasts, and megakaryocytes, but neutrophils or normoblasts did not stain for FasL (Fig. 2). Using VEGF ELISA, the mean cellular VEGF levels in newly diagnosed AML patients and controls were 32.199 25.21 (range 0.14– 98.95) and 15.309 13.80 pg/ mL (range 0.14–41.28), respectively, showing wide ranges of VEGF level. The difference was significant (P= 0.031).
3.2. Correlation between angiogenesis and the VEGF le6el or FasL expression in AML In newly diagnosed AML patients, there was a significant positive correlation between the MVC and the VEGF level (r= 0.43, P=0.025) (Fig. 3). This finding may support that both angiogenesis and VEGF, a potent angiogenic stimulator, interact via a paracrine pathway. To explore the relationship between tumor angiogenesis and tumor immune escape, we compared the MVC and VEGF level with the degree of FasL expression in patients with newly diagnosed AML. However, there was no correlation between FasL expression and the MVC (r= −0.113, P= 0.592) or between FasL expression and the VEGF level (r= −0.263, P= 0.133). These findings suggest that angiogenesis and FasL, considered as one of the mechanisms of immune escape in leukemia [12,16,17], are independent processes in newly diagnosed AML, although we did not perform further studies about apoptosis of tumor-specific cytotoxic T-cells.
3.3. Correlation between hematological or clinical parameters and the MVC, VEGF le6el, or FasL expression There was a positive correlation between the MVC and percentage of BM blasts (r= 0.409, P= 0.011) (Fig. 4). This finding suggests that the angiogenesis in AML may be a relationship with tumor burden. However, there were no correlations between the MVC and other hematologic parameters, including leukocyte count, hemoglobin level, platelet count, percentage of peripheral blood blasts, and BM cellularity. In addition, there were no correlations between the VEGF level or FasL expression and the hematologic parameters. BM specimens from 18 non-APL patients and eight APL patients, who underwent induction therapy with intense chemotherapy and an ATRA-based regimen, respectively, were stained immunohistochemically for vWF and FasL. Eleven patients (68.8%) with non-APL and seven patients (87.5%) with APL had achieved complete remission (CR) at the time of the BM exam after the first induction therapy. There were no correla-
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Fig. 1. Immunohistochemical staining ( × 400 field) of bone marrow (BM) sections from AML patients at diagnosis (A), after induction therapy (B), and controls (C) with antibodies for von Willebrand Factor (vWF). The microvessel count (MVC) in the AML marrow at diagnosis (A) is significantly greater than in controls (C). The BM MVC in AML patients after induction therapy (B) is reduced compared to newly diagnosed AML patients (A), but is higher than in controls (C). The cytoplasm of megakaryocytes stained strongly for vWF in controls and AML.
Fig. 2. Immunohistochemical staining ( × 400 field) of BM sections from AML patients at diagnosis (A), after induction therapy (B), and controls (C) with antibodies for Fas-ligand (FasL). The FasL expression in AML patients at diagnosis (A) was significantly higher than in controls (C). The FasL expression in BM from AML patient after induction therapy (B) is lower than in AML patients at diagnosis (A), but greater than in controls (C).
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Fig. 3. Correlation of MVC (No./× 400 field) with the VEGF level (pg/mL) in AML patients at diagnosis (r = 0.43, P =0.025).
tions between the presence of CR and the MVC or FasL expression. In addition, there were no significant differences between APL patients and non-APL patients for MVC, VEGF level, or FasL expression.
4. Discussion The role of angiogenesis in the growth of the primary tumor and metastatic lesion is well established in solid tumors [1– 3]. Without inducing angiogenesis, the tumors cannot grow beyond a size of 1– 2 mm3 [1,2]. In addition, solid tumors with higher angiogenesis as measured by the MVC or MVD have poorer clinical outcomes [2,3]. Recently, several studies, including this study, have reported significantly increased angiogenesis in the BM of patients with AML compared to normal controls, suggesting an important role of angio-
Fig. 4. Correlation of MVC (No./× 400 field) with the percentage of BM blasts (%) (r =0.409, P= 0.011).
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genesis in the pathogenesis or promotion of AML [25,26]. These findings suggest that angiogenic inhibitors may be a therapeutic modality in AML, as in solid tumors and multiple myeloma [2,27,28]. Actually, thalidomide has been tried as anti-angiogenesis therapy in patients with refractory or relapsed leukemia, with some clinical improvement [29]. Whether there is a correlation between angiogenesis and tumor burden in the BM of AML patients is somewhat controversial. Consistent with this study, Hussong et al. [25] reported that there was a positive correlation between the microvessel count and the percentage of BM blasts in newly diagnosed AML patients, whereas Padro et al. [26] disagreed. Similarly, in a study of MDS, refractory anemia with excess of blasts in transformation had a greater MVD than refractory anemia, refractory anemia with ringed sideroblasts, or refractory anemia with excess of blasts, while the MVD in AML was higher than in MDS; suggesting a correlation between angiogenesis and leukemic progression [30]. Further study with many more patients is needed to define the relationship between angiogenesis and tumor burden. Several chemotherapeutic agents, including anthracycline, vinca alkaloids, paclitaxel, bleomycin, titanocene, camptothecin, and topotecan, are anti-angiogenic in vitro and in vivo [31–35]. Recently, Kini et al. [36] reported that ATRA has an anti-angiogenesis effect by inhibiting VEGF production in leukemic cells in APL. As expected, our study found that angiogenesis was significantly reduced in AML patients who underwent induction therapy with anthracycline-based chemotherapy for non-APL or ATRA-based therapy for APL, compared to the level before therapy. In addition, we didn’t find any statistical difference in the angiogenesis between APL patients and non-APL patients. Padro et al. [26] reported that the range of MVD in BM taken during CR was the same as in controls. In this study, however, there was no difference in the MVC at diagnosis or at determination of response according to the presence of CR and angiogenesis after induction therapy was higher than in controls. The reasons for this discrepancy are not clear. Several factors, including the limited number of patients studied, variation in the subjective interpretation of immunohistochemical staining, and differences in the date of the BM study, should be considered. VEGF is a soluble, 34–46-kDa, heparin-binding glycoprotein dimer, with four different forms, i.e. VEGFA, B, C, and D [37]. VEGF-A, the most potent form of VEGF, exists in four isoforms (VEGF121, 165, 189, 206) by alternative splicing of mRNA [37]. In this study, we measured VEGF levels using ELISA to detect VEGF-A. The major known action of VEGF is as a potent endothelial mitogen that induces angiogenesis by stimulating endothelial proliferation and reducing endothelial death in solid tumors [1,2]. In hematopoietic
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malignancies, there is increasing evidence that angiogenic growth factors play important roles in angiogenesis via a paracrine pathway [5,9,10]. In addition, several hematopoietic cell lines are observed to express VEGF receptors, suggesting an autocrine pathway in which tumor cells may stimulate their own growth after VEGF exposure [9]. In this study, we observed a positive correlation between the MVC and VEGF level, suggesting that leukemic cells secrete angiogenic stimulators, which induce angiogenesis via a paracrine pathway. Recent serial studies [23,24] reported that VEGF inhibits apoptotic cell death in normal hematopoietic stem cell and leukemic cell line and this inhibitory effect of VEGF may contribute by secretion of ZK7 protein in these cells. FasL and its receptor, Fas (APO-1/CD95), play a crucial regulatory role in immune-mediated apoptosis [11,15]. FasL induces killing of Fas-sensitive cells, expressed on certain neoplastic and normal cells including activated T lymphocytes, through FasL-Fas interaction [11,12,15,38]. The expression of FasL was initially discovered in activated T-cells or NK cells, as well as in immune privileged sites, such as the testis or anterior ocular chamber [39]. Recently, several types of solid tumors were also found to express FasL [13]. The FasL-expressing tumor cells induce the killing of Fassensitive activated T-lymphocytes, the so-called Fas counterattack, resulting in tumor immune escape [13,14]. T-cells are known to play important roles in two aspects of hematology. One is that T-cell depletion in allogeneic BMT causes the high incidence of relapse [40]. The other is in donor lymphocyte infusion (DLI) used to treat relapsed leukemia after allogeneic BMT. The response rates of chronic myeloid leukemia (CML), AML, and acute lymphoblastic leukemia to DLI are 70, below 40, and 20%, respectively [41,42]. Buzyn et al. [17] reported that 54% of AML patients and 27% of CML patients have functional FasL activity, as assessed by the ability of leukemic cells to induce the apoptosis of Fas-sensitive target cells. Differences in FasL expression in leukemic cells may result in the different response rates for DLI, implicating leukemic immune escape through the Fas/FasL pathway. This has implications about which patients are eligible for adoptive cellular immunotherapy and further studies are needed urgently to define any clinical relationship between FasL expression and prediction of response to DLI. This study confirms that FasL expression in newly diagnosed AML patients is greater than in normal controls. In addition, as in angiogenesis, FasL expression after induction therapy is reduced compared to the expression at diagnosis, but some patients are still higher than in controls. These findings suggest that blockade of the Fas/FasL pathway may be used as a therapeutic modality. In solid tumor, somewhat contradictory results have been reported regarding relationship between angiogenic factors (VEGF and MVD) and apoptotic factors (FasL
and caspase-3) [20– 22]. In addition, the angiogenic factor inhibits apoptotic cell death in hematopoietic cells by producing ZK7 protein in vitro [23,24]. Since FasL is known as one of apoptotic factors and is involved in the mechanisms of tumor immune escapes, we evaluated the relationship between angiogenesis and FasL expression in AML. FasL expression in AML patients showed no correlation with the angiogenesis or the VEGF level. This suggests that angiogenesis and tumor immune escape in AML may contribute to leukemogenesis by independent processes. Our study has several methodological limitations in interpreting angiogenesis or tumor immune escape in patients with AML: first, the problem of subjective interpretation of immunohistochemical stainings for vWF or FasL may exist. Second, cellular VEGF levels were measured rather than secretory form due to unable serum samples. Third, some changes of T-cell related immune escape should be studied. In conclusion, our findings provide evidence of increased angiogenesis and tumor FasL expression in AML patients, and suggest that angiogenesis and FasL expression, tumor immune escape, are independent processes in AML. These findings may provide a rationale to suggest that angiogenesis inhibitors or blockade of the Fas/FasL pathway may be used as therapeutic modalities.
Acknowledgements The authors thank Dr Y. Takaue, Department of Medical Oncology, National Cancer Center Hospital, Japan for critical review during the preparation of the manuscript. J.-J. Lee provided the concept, design, assembled and analyzed the data, provided statistical expertise and drafted the paper. I.-J. Chung provided study materials and critical revision. M.-R. Park provided and collected the data. D-W. Ryang provided logistical support and helped with data interpretation. C.-S. Park obtained the necessary funding, provided administrative support and assisted with data analysis. H.-J. Kim provided critical revision and gave final approval.
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Increased angiogenesis and Fas-ligand expression are independent processes in acute myeloid leukemia Je-Jung Lee a, Ik-Joo Chung a, Moo-Rim Park a, Dong-Wook Ryang b, Chang-Soo Park c, Hyeoung-Joon Kim a,* a b
Department of Internal Medicine, Chonnam National Uni6ersity Medical School, 8 Hak-dong, Dong-ku, Kwangju 501 -757, South Korea Department of Clinical Pathology, Chonnam National Uni6ersity Medical School, 8 Hak-dong, Dong-ku, Kwangju 501 -757, South Korea c Department of Pathology, Chonnam National Uni6ersity Medical School, 8 Hak-dong, Dong-ku, Kwangju 501 -757, South Korea Received 10 May 2000; accepted 30 April 2001
Abstract We evaluated the clinical significance of tumor angiogenesis and Fas-ligand (FasL) expression using parameters including the microvessel count (MVC), vascular endothelial growth factor (VEGF) level, and FasL expression in patients with acute myeloid leukemia (AML). Paraffin-embedded bone marrow (BM) sections from 43 AML patients at diagnosis, 20 patients after subsequent induction therapy, and 18 controls with non-invasive lymphoma were stained immunohistochemically for von Willebrand factor (vWF) and FasL. VEGF in BM mononuclear cells from 32 AML patients at diagnosis and 10 controls, including bone marrow transplantation donors, was assayed by an ELISA method. We found that the mean MVC, VEGF level, and FasL expression in AML patients at diagnosis were significantly higher than those of controls, with a significant correlation between the MVC and VEGF levels (r= 0.43). However, there were no correlations between FasL expression and MVC or VEGF level. The mean MVC and FasL expression after induction therapy were lower than those evaluated at diagnosis, but were higher than those of controls. There was a correlation between the MVC and percentage of BM blasts (r= 0.479), but no correlation between the MVC, VEGF level, or FasL expression and other hematologic or clinical variables. Our findings provide evidence of increased angiogenesis and tumor immune escape in AML, and both angiogenesis and tumor immune escape are independent processes in AML. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Microvessel count; VEGF; Tumor immune escape; Fas-ligand; AML
1. Introduction Angiogenesis plays an important role in the growth of primary tumors and metastatic sites of solid tumors, and is an independent clinical prognostic factor in Abbre6iations: AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ara-C, cytarabine; ATRA, all-trans retinoic acid; BM, bone marrow; BMT, bone marrow transplantation; CML, chronic myeloid leukemia; CR, complete remission; DLI, donor lymphocyte infusion; FAB, French–American–British; FasL, Fas-ligand; IDA, idarubicin; MDS, myelodysplastic syndrome; NK, natural killer; MNC, mononuclear cell; MVC, microvessel count; MVD, microvessel density; SD, standard deviation; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor. * Corresponding author. Tel.: +82-62-2206-572; fax: + 82-622258-578. E-mail address: [email protected] (H.-J. Kim).
several malignancies [1–3]. A balance between angiogenic stimulators and angiogenesis inhibitors tightly regulates angiogenesis in endothelial cells [1,4]. Of the proteins known to activate endothelial growth and movement, vascular endothelial growth factor (VEGF) seem to be one of the most potent mitogens for endothelial cells in the neovascularization of solid tumors and is associated with enhanced angiogenesis, metastasis, and poor outcome in solid tumors [1,2]. Recently, several investigators evaluated angiogenesis and VEGF in hematologic malignancies, such as multiple myeloma, myelodysplastic syndrome (MDS), leukemia, and lymphoma, but its exact role in pathogenesis is still unclear [5–10]. Fas-ligand (FasL) is a 37-kDa transmembrane protein that induces apoptosis by killing Fas-positive target cells through both juxtacrine and paracrine
0145-2126/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(01)00082-0
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mechanisms [11,12]. Recently, FasL expression was found in several types of solid tumors or tumor cell lines [13]. Abnormal FasL expression in these tumor cells induces the killing of activated tumor-specific Tcells or natural killer (NK) cells bearing Fas at the tumor site, resulting in escape from tumor immune surveillance [13,14]. Recently, some investigators have reported that leukemic cells also have a mechanism of tumor immune escape involving the Fas/FasL pathway [12,15– 17]. The relationship and balance between angiogenesis and apoptosis in growth of solid tumors is still unclear. Hypoxia, which is a potent stimulator of angiogenesis, is closely correlated with factors known to induce apoptosis [18,19]. Based on this finding, some investigators reported an inverse correlation between angiogenic factors and apoptotic factors in gastric and lung cancer [20,21], but any correlation was denied in breast cancer [22]. In hematopoietic cells, Katoh et al. [23,24] reported that VEGF inhibits apoptotic cell death by producing ZK7 protein in vitro. However, direct comparison between angiogeneic factors and FasL expression, one of the mechanisms for tumor immune escape, has not been extensively studied in acute myeloid leukemia (AML). In this study, we evaluated the clinical significance of angiogenesis, VEGF, and FasL expression in patients with AML.
2. Materials and methods
idarubicin (IDA) (12 mg/m2/day IV over 30 minutes on days 1–3) and cytarabine (ara-C) (100 mg/m2/day IV continuously on days 1–7) for non-APL and all-trans retinoic acid (ATRA) (45 mg/m2 orally until complete remission) and/or IDA (12 mg/m2/day IV over 30 min on days 2, 4, 6, and 8) for APL.
2.2. Immunohistochemistry We used the avidin-biotin-peroxidase complex method of immunohistochemical staining with a Microprobe Immuno/DNA stainer (Fisher Scientific, Pittsburgh, PA). Three-mm-thick BM sections were mounted on slides and deparaffinized. For FasL staining, the sections were heated in a microwave oven in 2.1% citric acid buffer solution (pH 6.0) for 7 min to retrieve the antigens. For vWF staining, sections were incubated with Pepsin solution (Research Genetics, Huntsville, AL) for 3 min at 45 °C. After preincubation with 0.6% hydrogen peroxide, protein blocker, and anti-biotin for 5 min at 45 °C, the sections were incubated in the primary antibody for vWF (1:500 working concentration) for 15 min at 45 °C or FasL (1:100 working concentration) for 90 min at room temperature in a moist chamber. After washing, the sections were incubated in biotinylated anti-mouse IgG (Sigma, St Louis, MO) for 7 min at 45 °C. The streptavidin-horseradish peroxide detection system (Research Genetics) was applied for 7 min at 45 °C. The sections were developed with the chromogen 3-amino-9-ethylcarbazole (AEC) for 7 min at 45 °C. Counter staining was performed with hematoxylin for 1 min at 45 °C.
2.1. Patients 2.3. Determination of micro6essel count (MVC) Paraffin-embedded bone marrow (BM) sections from 43 patients with newly diagnosed AML and 20 patients who received remission induction therapy at Chonnam National University Hospital from May 1999 to January 2000 were stained immunohistochemically for von Willebrand factor (vWF) (Dako, Carpinteria, CA) and FasL (Santa Cruz Biotechnology, Santa Cruz, CA). Control specimens were left over biopsed samples from 18 patients with lymphoma for staging with normal BM morphology. In addition, BM mononuclear cells (MNCs) from 32 patients with newly diagnosed AML were assayed for VEGF by an ELISA method (R&D Systems, Minneapolis, MN). The controls for VEGF were MNCs from 10 healthy donors for allogeneic bone marrow transplantation (BMT). The median age of the patients was 45 years (range 16–78). There were 24 males and 19 females. According to the French–American – British (FAB) classification, 10 patients had acute promyelocytic leukemia (APL) and 33 patients had non-APL. Of 43 patients, 18 patients with non-APL and eight patients with APL received remission induction therapy, which consisted of
After selecting the two areas with the highest microvessel density (MVD) on a ×200 field, the MVC was assessed in ×400 fields. Areas of staining with no discrete breaks were counted as a single vessel. Large vessels (] 10 mL) or vessels in the periosteum or bone were excluded.
2.4. VEGF ELISA BM MNCs were separated by centrifugation using Ficoll-Hypague (Lymphoprep™, Nycomed, Oslo, Norway). The cells were sonicated with three 15-s, 60 W-bursts of a Fisher sonic dismembrator, and subsequently clarified by centrifugation at 12,000 rpm for 10 min at 4 °C. For each sample, 50 mg of protein measured with the BCA protein assay kit were tested using sandwich ELISA according to the manufacturer’s instruction (R&D Systems, USA), and all analyses and calibrations were carried out in duplicate. Samples (200 mL) and standards (200 mL) were added to a coated microtiter plate and incubated for 2 h at room temper-
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ature. After washing with buffer solution three times, 200 mL of recombinant anti-VEGF polyclonal antibody conjugated to horseradish peroxide were added to the wells. After an additional 2-h incubation, the wells were rinsed again, and 200 mL of hydrogen peroxide and tetramethylbenzidine were added. The reaction was stopped by the addition of 50 mL of 2 N sulfuric acid. The absorbance of each well was measured using a microplate reader at 450 nm and plotted against a standard curve with VEGF levels expressed in pg/mL.
2.5. Statistical analysis All statistical analyses were performed with the program SPSS 10.0 (SPSS, Chicago, IL). The MVC, VEGF level, and FasL expression of the patients and controls were compared using the Mann-Whitney U-test or the Kruskal-Wallis test. Correlations between clinical or hematological variables and MVC, VEGF level, or FasL expression used Pearson’s test for parametric variables and Spearman’s z test for nonparametric variables. P-valuesB 0.05 were considered statistically significant. The results are expressed as the mean plus or minus the standard deviation (SD).
3. Results
3.1. MVC, VEGF le6el, and FasL expression are increased in AML Using vWF staining (Fig. 1), the mean MVC was 31.99 14.3 vessels/ × 400 field (range 9-71) in newly diagnosed AML patients, 18.697.9 vessels/ × 400 field (range 6–35) in patients after induction therapy, and 9.1 9 2.8 vessels/ × 400 field (range 4– 14) in controls. The MVC in patients with newly diagnosed AML was significantly higher than in controls (PB 0.001). The mean MVC in patients after induction therapy was significantly lower than that of newly diagnosed AML patients (PB 0.001), but was still higher than that of controls (PB0.001). However, patients with newly diagnosed AML showed wide ranges of the MVC. In addition, the cytoplasm of megakaryocytes stained strongly for vWF (Fig. 1). With FasL staining (Fig. 2), the mean expression of FasL in newly diagnosed AML patients, patients after induction therapy, and controls was 55.19 27.2 (range 5–95), 39.3924.4 (range 10– 85), and 12.89 10.3% (range 3–35), respectively. The differences between the expression of FasL in newly diagnosed AML patients and controls were highly significant (P B 0.001). The mean expression of FasL in patients after induction therapy was lower than that of newly diagnosed AML patients (P= 0.037). Although the mean expression of FasL in patients after induction therapy was still higher
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than that of controls (PB 0.001), many patients underwent induction therapy showed decreased FasL expression. However, there were wide ranges of FasL expression within each group. The FasL staining was positive in lymphocytes, myeloblasts, erythroblasts, and megakaryocytes, but neutrophils or normoblasts did not stain for FasL (Fig. 2). Using VEGF ELISA, the mean cellular VEGF levels in newly diagnosed AML patients and controls were 32.199 25.21 (range 0.14– 98.95) and 15.309 13.80 pg/ mL (range 0.14–41.28), respectively, showing wide ranges of VEGF level. The difference was significant (P= 0.031).
3.2. Correlation between angiogenesis and the VEGF le6el or FasL expression in AML In newly diagnosed AML patients, there was a significant positive correlation between the MVC and the VEGF level (r= 0.43, P=0.025) (Fig. 3). This finding may support that both angiogenesis and VEGF, a potent angiogenic stimulator, interact via a paracrine pathway. To explore the relationship between tumor angiogenesis and tumor immune escape, we compared the MVC and VEGF level with the degree of FasL expression in patients with newly diagnosed AML. However, there was no correlation between FasL expression and the MVC (r= −0.113, P= 0.592) or between FasL expression and the VEGF level (r= −0.263, P= 0.133). These findings suggest that angiogenesis and FasL, considered as one of the mechanisms of immune escape in leukemia [12,16,17], are independent processes in newly diagnosed AML, although we did not perform further studies about apoptosis of tumor-specific cytotoxic T-cells.
3.3. Correlation between hematological or clinical parameters and the MVC, VEGF le6el, or FasL expression There was a positive correlation between the MVC and percentage of BM blasts (r= 0.409, P= 0.011) (Fig. 4). This finding suggests that the angiogenesis in AML may be a relationship with tumor burden. However, there were no correlations between the MVC and other hematologic parameters, including leukocyte count, hemoglobin level, platelet count, percentage of peripheral blood blasts, and BM cellularity. In addition, there were no correlations between the VEGF level or FasL expression and the hematologic parameters. BM specimens from 18 non-APL patients and eight APL patients, who underwent induction therapy with intense chemotherapy and an ATRA-based regimen, respectively, were stained immunohistochemically for vWF and FasL. Eleven patients (68.8%) with non-APL and seven patients (87.5%) with APL had achieved complete remission (CR) at the time of the BM exam after the first induction therapy. There were no correla-
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Fig. 1. Immunohistochemical staining ( × 400 field) of bone marrow (BM) sections from AML patients at diagnosis (A), after induction therapy (B), and controls (C) with antibodies for von Willebrand Factor (vWF). The microvessel count (MVC) in the AML marrow at diagnosis (A) is significantly greater than in controls (C). The BM MVC in AML patients after induction therapy (B) is reduced compared to newly diagnosed AML patients (A), but is higher than in controls (C). The cytoplasm of megakaryocytes stained strongly for vWF in controls and AML.
Fig. 2. Immunohistochemical staining ( × 400 field) of BM sections from AML patients at diagnosis (A), after induction therapy (B), and controls (C) with antibodies for Fas-ligand (FasL). The FasL expression in AML patients at diagnosis (A) was significantly higher than in controls (C). The FasL expression in BM from AML patient after induction therapy (B) is lower than in AML patients at diagnosis (A), but greater than in controls (C).
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Fig. 3. Correlation of MVC (No./× 400 field) with the VEGF level (pg/mL) in AML patients at diagnosis (r = 0.43, P =0.025).
tions between the presence of CR and the MVC or FasL expression. In addition, there were no significant differences between APL patients and non-APL patients for MVC, VEGF level, or FasL expression.
4. Discussion The role of angiogenesis in the growth of the primary tumor and metastatic lesion is well established in solid tumors [1– 3]. Without inducing angiogenesis, the tumors cannot grow beyond a size of 1– 2 mm3 [1,2]. In addition, solid tumors with higher angiogenesis as measured by the MVC or MVD have poorer clinical outcomes [2,3]. Recently, several studies, including this study, have reported significantly increased angiogenesis in the BM of patients with AML compared to normal controls, suggesting an important role of angio-
Fig. 4. Correlation of MVC (No./× 400 field) with the percentage of BM blasts (%) (r =0.409, P= 0.011).
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genesis in the pathogenesis or promotion of AML [25,26]. These findings suggest that angiogenic inhibitors may be a therapeutic modality in AML, as in solid tumors and multiple myeloma [2,27,28]. Actually, thalidomide has been tried as anti-angiogenesis therapy in patients with refractory or relapsed leukemia, with some clinical improvement [29]. Whether there is a correlation between angiogenesis and tumor burden in the BM of AML patients is somewhat controversial. Consistent with this study, Hussong et al. [25] reported that there was a positive correlation between the microvessel count and the percentage of BM blasts in newly diagnosed AML patients, whereas Padro et al. [26] disagreed. Similarly, in a study of MDS, refractory anemia with excess of blasts in transformation had a greater MVD than refractory anemia, refractory anemia with ringed sideroblasts, or refractory anemia with excess of blasts, while the MVD in AML was higher than in MDS; suggesting a correlation between angiogenesis and leukemic progression [30]. Further study with many more patients is needed to define the relationship between angiogenesis and tumor burden. Several chemotherapeutic agents, including anthracycline, vinca alkaloids, paclitaxel, bleomycin, titanocene, camptothecin, and topotecan, are anti-angiogenic in vitro and in vivo [31–35]. Recently, Kini et al. [36] reported that ATRA has an anti-angiogenesis effect by inhibiting VEGF production in leukemic cells in APL. As expected, our study found that angiogenesis was significantly reduced in AML patients who underwent induction therapy with anthracycline-based chemotherapy for non-APL or ATRA-based therapy for APL, compared to the level before therapy. In addition, we didn’t find any statistical difference in the angiogenesis between APL patients and non-APL patients. Padro et al. [26] reported that the range of MVD in BM taken during CR was the same as in controls. In this study, however, there was no difference in the MVC at diagnosis or at determination of response according to the presence of CR and angiogenesis after induction therapy was higher than in controls. The reasons for this discrepancy are not clear. Several factors, including the limited number of patients studied, variation in the subjective interpretation of immunohistochemical staining, and differences in the date of the BM study, should be considered. VEGF is a soluble, 34–46-kDa, heparin-binding glycoprotein dimer, with four different forms, i.e. VEGFA, B, C, and D [37]. VEGF-A, the most potent form of VEGF, exists in four isoforms (VEGF121, 165, 189, 206) by alternative splicing of mRNA [37]. In this study, we measured VEGF levels using ELISA to detect VEGF-A. The major known action of VEGF is as a potent endothelial mitogen that induces angiogenesis by stimulating endothelial proliferation and reducing endothelial death in solid tumors [1,2]. In hematopoietic
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malignancies, there is increasing evidence that angiogenic growth factors play important roles in angiogenesis via a paracrine pathway [5,9,10]. In addition, several hematopoietic cell lines are observed to express VEGF receptors, suggesting an autocrine pathway in which tumor cells may stimulate their own growth after VEGF exposure [9]. In this study, we observed a positive correlation between the MVC and VEGF level, suggesting that leukemic cells secrete angiogenic stimulators, which induce angiogenesis via a paracrine pathway. Recent serial studies [23,24] reported that VEGF inhibits apoptotic cell death in normal hematopoietic stem cell and leukemic cell line and this inhibitory effect of VEGF may contribute by secretion of ZK7 protein in these cells. FasL and its receptor, Fas (APO-1/CD95), play a crucial regulatory role in immune-mediated apoptosis [11,15]. FasL induces killing of Fas-sensitive cells, expressed on certain neoplastic and normal cells including activated T lymphocytes, through FasL-Fas interaction [11,12,15,38]. The expression of FasL was initially discovered in activated T-cells or NK cells, as well as in immune privileged sites, such as the testis or anterior ocular chamber [39]. Recently, several types of solid tumors were also found to express FasL [13]. The FasL-expressing tumor cells induce the killing of Fassensitive activated T-lymphocytes, the so-called Fas counterattack, resulting in tumor immune escape [13,14]. T-cells are known to play important roles in two aspects of hematology. One is that T-cell depletion in allogeneic BMT causes the high incidence of relapse [40]. The other is in donor lymphocyte infusion (DLI) used to treat relapsed leukemia after allogeneic BMT. The response rates of chronic myeloid leukemia (CML), AML, and acute lymphoblastic leukemia to DLI are 70, below 40, and 20%, respectively [41,42]. Buzyn et al. [17] reported that 54% of AML patients and 27% of CML patients have functional FasL activity, as assessed by the ability of leukemic cells to induce the apoptosis of Fas-sensitive target cells. Differences in FasL expression in leukemic cells may result in the different response rates for DLI, implicating leukemic immune escape through the Fas/FasL pathway. This has implications about which patients are eligible for adoptive cellular immunotherapy and further studies are needed urgently to define any clinical relationship between FasL expression and prediction of response to DLI. This study confirms that FasL expression in newly diagnosed AML patients is greater than in normal controls. In addition, as in angiogenesis, FasL expression after induction therapy is reduced compared to the expression at diagnosis, but some patients are still higher than in controls. These findings suggest that blockade of the Fas/FasL pathway may be used as a therapeutic modality. In solid tumor, somewhat contradictory results have been reported regarding relationship between angiogenic factors (VEGF and MVD) and apoptotic factors (FasL
and caspase-3) [20– 22]. In addition, the angiogenic factor inhibits apoptotic cell death in hematopoietic cells by producing ZK7 protein in vitro [23,24]. Since FasL is known as one of apoptotic factors and is involved in the mechanisms of tumor immune escapes, we evaluated the relationship between angiogenesis and FasL expression in AML. FasL expression in AML patients showed no correlation with the angiogenesis or the VEGF level. This suggests that angiogenesis and tumor immune escape in AML may contribute to leukemogenesis by independent processes. Our study has several methodological limitations in interpreting angiogenesis or tumor immune escape in patients with AML: first, the problem of subjective interpretation of immunohistochemical stainings for vWF or FasL may exist. Second, cellular VEGF levels were measured rather than secretory form due to unable serum samples. Third, some changes of T-cell related immune escape should be studied. In conclusion, our findings provide evidence of increased angiogenesis and tumor FasL expression in AML patients, and suggest that angiogenesis and FasL expression, tumor immune escape, are independent processes in AML. These findings may provide a rationale to suggest that angiogenesis inhibitors or blockade of the Fas/FasL pathway may be used as therapeutic modalities.
Acknowledgements The authors thank Dr Y. Takaue, Department of Medical Oncology, National Cancer Center Hospital, Japan for critical review during the preparation of the manuscript. J.-J. Lee provided the concept, design, assembled and analyzed the data, provided statistical expertise and drafted the paper. I.-J. Chung provided study materials and critical revision. M.-R. Park provided and collected the data. D-W. Ryang provided logistical support and helped with data interpretation. C.-S. Park obtained the necessary funding, provided administrative support and assisted with data analysis. H.-J. Kim provided critical revision and gave final approval.
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