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Journal of Clinical Oncology, Vol 23, No 6 (February 20), 2005: pp. 1109-1117 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.05.058 Analysis of Concerted Expression of Angiogenic Growth Factors in Acute Myeloid Leukemia: Expression of Angiopoietin-2 Represents an Independent Prognostic Factor for Overall SurvivalFrom the Medizinische Klinik II, University Hospital Hamburg-Eppendorf; Institute of Medical Biometry and Epidemiology, University Hospital Hamburg-Eppendorf, Hamburg; and Department of Oncology/Hematology, Medical University Hannover, Hannover, Germany Address reprint requests to Walter Fiedler, MD, Medizinische Klinik II, University Hospital Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; e-mail: fiedler{at}uke.uni-hamburg.de
PURPOSE: Bone marrow neoangiogenesis plays an important pathogenetic and possible prognostic role in acute myeloid leukemia (AML). Members of the vascular endothelial growth factor (VEGF) and angiopoietin family represent the most specific inducers of angiogenesis secreted by AML blasts. We therefore correlated expression of angiogenic factors with clinical variables. PATIENTS AND METHODS: We investigated the expression of VEGF-A, VEGF-C, angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), and the receptor Tie2 by quantitative polymerase chain reaction in a cohort of 90 patients younger than 61 years with de novo AML entered into the German AML Süddeutsche Hämoblastose Gruppe Hannover 95 trial. Uni- and multivariate analyses were performed using clinical and gene expression variables. RESULTS: Univariate analysis of overall survival indicated the following variables as prognostic factors: good response on a day-15 bone marrow examination after initiation of induction chemotherapy, karyotype, and high Ang2 expression. In multivariate analysis, only bad response and log Ang2 expression remained of statistical significance, with a hazard ratio of 3.51 (95% CI, 1.91 to 6.47) and 0.75 (95% CI, 0.61 to 0.91), respectively. Subgroup analysis suggested that the prognostic impact of Ang2 expression was especially evident in cohorts with low VEGF-C and Ang1 mRNA levels. CONCLUSION: These results show that expression of Ang2 represents an independent prognostic factor in AML. Additional research into interactions of angiogenic cytokines in the pathogenesis of bone marrow angiogenesis in AML is warranted.
Neoangiogenesis plays a crucial role in the growth of solid tumors and of hematologic malignancies such as acute myeloid leukemia (AML).1,2 Increased numbers of endothelial cells have been detected on histologic sections of bone marrow biopsies of AML patients compared with those with reactive disorders. Paracrine exchange of growth factors between AML blasts and endothelial cells is believed to contribute to the pathogenesis of AML.3 Although many endothelial mitogens have been identified, most are nonselective (eg, fibroblast growth factor 2 stimulates proliferation of different cell types).4–6 The two major classes of endothelium-specific growth factors include the vascular endothelial growth factor (VEGF) and the angiopoietin family. Known members of the VEGF family are VEGF-A, -B, -C, -D, -E, and placental growth factor. The angiopoietin family comprises Ang1, 2, 3, and 4. There are three known VEGF receptors (VEGF receptor 1, 2, and 3)7 and one angiopoietin receptor (Tie2).8,9 Both classes of receptors belong to the tyrosine kinase family and are exclusively expressed on endothelial cells, hematopoietic stem cells, and tumor cells.10–13 Interactions between members of the VEGF and angiopoietin family are necessary for efficient growth and remodeling of blood vessels.14–16 In AML, bone marrow neoangiogenesis is promoted by growth factors released by leukemic blasts. Our group and others demonstrated that constitutive secretion of VEGF-A by AML blasts occurred in 71% of analyzed patients.17,18 Aguayo et al19 showed that VEGF-A expression represents a negative prognostic factor in patients with AML. Furthermore, we reported the expression of VEGF-C and its receptor Flt-4 by leukemic cells in 61% and 36% of AML patients, respectively.20 Dias et al21 recently found that VEGF-C released from the bone marrow endothelium induced proliferation, promoted survival, and protected leukemic cells from chemotherapy-induced apoptosis. Expression of angiopoietins and their receptor Tie2 by AML cells was studied by two groups. Muller et al22 described RNA expression of Ang1 and Tie2 in 11 of 17 patients with acute or chronic myeloid leukemia. Watarai et al23 reported a significantly higher expression of Ang2 by CD7+ AML blasts compared with those with the translocation t(8,21), suggesting a cell- and karyotype-specific expression. Given that neoangiogenesis depends on the interplay of different members of the VEGF and angiopoietin family, it is mandatory to determine their expression in the same pretreatment samples. In this study, we investigated the concerted expression of VEGF-A, VEGF-C, Ang1, Ang2, and Tie2 by quantitative polymerase chain reaction (PCR) in 90 patients younger than 61 years of age with de novo AML treated according to a uniform chemotherapy protocol. Uni- and multivariate analyses were performed to identify the prognostic impact of the expression of angiogenic factors on the long-term prognosis of AML patients.
Patients and Samples We analyzed peripheral-blood mononuclear cells (PBMCs) from 90 leukemic patients between 18 and 60 years of age with de novo AML. Patients belonged to a German multicenter clinical trial (AML Süddeutsche Hämoblastose Gruppe Hannover) onto which 305 patients had been entered between March 1995 and June 1999.24 Patients with t(15;17) were excluded from the trial. In brief, the trial design was as follows. Patients received induction chemotherapy with cytarabine, idarubicin, and etoposide. Karyotype and response on a day-15 bone marrow were used for additional stratification. Patients with normal karyotype, t(8;21), or inv(16) and a good response on day 15 (< 5% blasts in a hypoplastic marrow) received a second induction cycle with the same drugs. All others had therapy with intermediate-dose cytarabine and amsacrine. Consolidation I for the good-risk group consisted of medium-dose cytarabine and daunorubicin; high-risk patients received a second cycle cytarabine and amsacrine. Patients without t(8;21) or inv(16) and a family marrow donor received an allotransplantation. For the second consolidation, the remaining patients received either an autograft or a cycle of high-dose cytarabine plus daunorubicin. For patients with alterations of chromosome 5 or 7, matched unrelated donor transplantation was permitted. From the entire study cohort, 90 samples had been submitted to the coordinating study center in Hannover where mononuclear cells had been isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. cDNA had been prepared and stored. Table 1 lists patient characteristics.
The study was approved by the ethics committees at each study site and was conducted in accordance with German drug development regulations and the Declaration of Helsinki.
Isolation of PBMCs and Preparation of AC133+ Cells From Leukapheresis Products
Isolation of RNA and Synthesis of cDNA
Primers for Real-Time PCR The VEGF primer was constructed to amplify the three splice variants of VEGF (VEGF165, VEGF121, and VEGF18926). The Ang1 primers were designed to amplify all known splice variants of Ang1.27 All primer sets had an efficiency of at least 1.81 determined by log dilutions of plasmid standards.
Real-Time Quantitative PCR Results of two independent analyses for each gene and sample or plasmid dilution were averaged. The calculated amount of the target genes was normalized to the housekeeping gene GAPDH. All data are presented as ratio of the target gene/GAPDH. Primer sequences and PCR conditions are available on request.
Cell Lines and Ang2 Protein Determination
Statistical Methods
As primary end points for survival analysis, we defined overall survival (starting from time of diagnosis) and relapse-free survival after remission (including only patients with remission, starting from remission time and defining death or relapse as an event). To investigate the relationship between gene expression variables, Spearman's Univariate survival analysis included Kaplan-Meier curves and log-rank tests. In addition, an multiple Cox proportional hazards regression analysis was performed (including construction of Martingale residual plots and verification of the proportional hazards assumption).28,29 All statistical analyses were done with SPSS 11.0 (SPSS Inc, Chicago, IL) and S-Plus 6.0 (Insightful Corp, Seattle, WA).
To assess the prognostic impact of the expression of angiogenic growth factors and the receptor Tie2, leukemic cells from 90 patients with de novo AML were studied. The samples were obtained from patients entered onto the German AML SHG Hannover 95 trial.24 All available cDNA samples from the entire study group were included in this investigation. Patients were treated according to a uniform chemotherapy protocol. The investigated subgroup and the complementary group were not significantly different: age 43.6 v 42.4 years (P = .63), male sex 43.3% v 54.5% (P = .08), favorable karyotype 66.2% v 73.9% (P = .49), good response to first induction chemotherapy 72.2% v 70.2% (P = .27), and complete remission (CR) rate 71.1% v 77.9% (P = .63). Median relapse-free survival (RFS) and overall survival (OS) of the analyzed group were 18.9 and 32.8 months, respectively. Patient characteristics are listed in Table 1. Median follow-up time of censored patients (n = 47) was 35.6 months. Median RFS and OS of the entire SHG AML study population were 23.4 and 31.7 months, respectively. Although RFS was slightly shorter in the analyzed subgroup, treatment outcome was not statistically significantly different (log-rank test OS, P = .92; RFS, P = .81). Samples from 90 AML patients, AC133+ cells from peripheral blood from seven different leukapheresis products, and PBMCs from four healthy donors were analyzed by real-time qPCR. We quantified the expression of VEGF-A, VEGF-C, Ang1, Ang2, and Tie2 as a ratio with the housekeeping gene GAPDH (Fig 1; Table 2). Median levels of VEGF-A, VEGF-C, Ang1, and Ang2 were higher in leukemic blasts than in normal peripheral-blood AC133+ cells. With the exception of VEGF-C, expression of analyzed angiogenic factors was low in normal PBMCs.
To investigate the relation between Ang2 mRNA levels and protein secretion, we determined concomitantly the expression of Ang2 in five human leukemic cell lines (TF-1, Mo7e, HL60, U937, and K562) by qPCR and by ELISA of cell culture supernatants. A correlation of 0.8 between mRNA and protein expression was found. Because of the wide range of individual data points, we used logarithms of values shifted by 1 for further analysis and graphical display. mRNA specific for VEGF-A was detected in 90%, mRNA specific for VEGF-C was detected in 73%, mRNA specific for Ang1 was detected in 96%, mRNA specific for Ang2 was detected in 98%, and mRNA specific for Tie2 was detected in 60% of patients. We performed univariate analyses to correlate gene expression with confounding factors age, sex, karyotype, and response on day 15 after induction chemotherapy with long-term outcome. Patients with less than 5% blasts in hypoplastic bone marrows 15 days after initiation of chemotherapy without evidence of extramedullary disease were considered as good responders. The karyotype of patients was initially categorized into three risk groups: t(8;21), inv(16); normal karyotype; and trisomy 8, abnormalities of chromosome 5, 7, 11, or complex abnormalities. Although observed CR rates varied among these groups with 90.9%, 73.5%, and 60.7%, respectively, we could not detect a statistically significant difference. Because in our cohort no significant survival difference was detected between patients with core-binding factor leukemias (n = 11) and those with normal karyotype (n = 50), both groups were combined for additional analysis. Comparable outcomes for patients with CBF leukemias and those with normal karyotype were also found in the entire Hannover 95 study cohort.24 For additional analysis, patients were divided into three groups of approximately equal size (VEGF-A < 3, 3 to 20, and > 20; Ang1 < 0.5, 0.5 to 1.5, and > 1.5; Ang2 < 2, 2 to 15, and >15). Given that only 60% and 73% of patients expressed VEGF-C and Tie2 respectively, patients were analyzed in two groups: zero and more than zero. First we analyzed whether a correlation existed between gene expression levels of individual angiogenic factors and blast count, age, sex, karyotype, French-American-British classification type, and good or bad response to induction chemotherapy; no association was detected. In particular, no relevant correlation between absolute or relative blast count in peripheral blood and expression of angiogenic factors could be detected (absolute value of Spearman's r < 0.3 for all angiogenic factors and particularly r < 0.1 for Ang2). In addition, for Ang2, mean blast counts were not different among our specified groups. Univariate analysis was then performed to identify factors that could predict the achievement of a CR. In our patient group, the only variable that correlated with obtaining a CR was good response to induction chemotherapy assessed on day 15 (Fisher's exact test, P < .001). Although the age of the entire group of patients was younger than 61 years at diagnosis, there was a trend that younger patients had higher CR rates (P = .059). No impact of the variables sex (P = .64), karyotype (P = .1), VEGF-A (P = .59), VEGF-C (P = .42), Ang1 (P = .08), Ang2 (P = .28), and Tie2 (P = 1.0) was found. Univariate analysis of factors associated with OS showed a better prognosis for patients with good response to induction chemotherapy (log-rank P < .001), favorable karyotype (P = .07), and high Ang2 expression (P = .005). We could not detect a prognostic impact on survival for the factors sex (P = .52), age (P = .69), VEGF-A (P = .72), VEGF-C (P = .32), Ang1 (P = .88), and Tie2 (P = .43). Results of univariate analysis are listed in Table 3. Figure 2 shows the Kaplan-Meier curves for survival stratified for good response and for Ang2. It should be noted that patients were grouped into cohorts of approximately equal size to enable us to illustrate the results by Kaplan-Meier curves. Because Cox regression and Martingale plots revealed a linear relation between log Ang2 and risk (described in this section), no clinically relevant cut-offs could be identified and arbitrary cut-offs were chosen.
We used the Cox proportional hazards model based on all 90 patients to perform multivariate analyses of the variables with a P value of less than .1 (karyotype, good or bad response, and Ang2 expression) on overall survival (stepwise procedure). In this analysis, log Ang2 was used as a continuous predictor variable. We could identify Ang2 expression and good versus bad response as independent prognostic factors for survival (Table 4). Response on day 15 was closely related to karyotype, as can be deduced by cross-table analysis (P = .005). In our cohort among 64 patients with good response, 83% possessed a favorable karyotype, whereas among the patients with bad response only 50% had favorable cytogenetics. Therefore, karyotype lost its prognostic significance in multivariate analysis in our cohort.
The Martingale residual plot after inclusion of the variable response indicates that the effect of Ang2 on survival could be adequately modeled by a linear term on the log scale (compare Loess smoothing in Fig 3).
The resulting hazard ratio for bad versus good response was 3.51 (95% CI, 1.91 to 6.47; P < .001) and the hazard ratio for log Ang2 was 0.75 (95% CI, 0.61 to 0.91; P = .005). Karyotype did not show an additional independent effect on survival in multivariate analysis (P = .19). Results of the Cox regression therefore coincide with results of univariate analysis (compare with Fig 2). The Martingale residual plot might also suggest the presence of four outliers. However, because the omission of these four patients (all of them had a poor response and did not die during time of investigation, probably due to allogeneic bone marrow transplantation in three of them) did not change coefficients and SEs in a noteworthy way, we chose to continue using the model including all 90 patients. In addition, we investigated the adequacy of the proportional hazards assumption of the final model by fitting a model including a time-dependent covariate for each variable. P values of .48 and .49 for these coefficients suggested no relevant time dependency. Furthermore, data were reanalyzed after censoring patients with allogeneic bone marrow transplantation at the time point of stem-cell infusion. No substantial difference in results was found. In univariate analysis karyotype seemed to be of more pronounced prognostic value (P = .0039). However, in multivariate analysis there was again no additional significant effect of karyotype (P = .19), which is consistent with the original model without censoring of patients who received bone marrow transplantation. To further illustrate the impact of good and bad response and level of Ang2 expression on overall survival, Kaplan-Meier plots of patients with good and bad response stratified for Ang2 expression in three groups are shown in Figure 4.
To investigate possible interactions between the expression of various angiogenic growth factors on patient survival, subgroup analysis was performed. Patients were divided into groups with expression of VEGF-A, VEGF-C, and Ang1 below or above the median. Survival estimates were calculated for each subgroup according to the level of Ang2 expression. Kaplan-Meier survival curves for all subgroups are shown in Figure 5. The prognostic relevance of Ang2 expression was most pronounced in the subgroups with low VEGF-C and Ang1 mRNA levels. In the cohorts with high VEGF-C, VEGF-A, and Ang1, Ang2 expression lost its impact on OS, suggesting that the protective effect of Ang2 is diminished by simultaneous expression of proangiogenic factors.
Statistical analysis of RFS was nonyielding, given that 26 patients did not achieve a complete remission. Because of low patient numbers and consequently lack of statistical power, no additional analysis was performed for RFS.
Bone marrow neoangiogenesis in AML is a complex process involving the interplay of different angiogenic growth factors. Until now, most investigators tried to elucidate the impact of single angiogenic factors on the pathogenesis or prognosis in hematologic malignancies and solid tumors.17,19,21,30–34 However, because of the orchestrated action of various angiogenic cytokines, it is necessary to analyze simultaneously their expression in identical pretreatment samples to gain insights into their relative relevance for the disease process. The endothelium-specific growth factors of the VEGF and angiopoietin family are believed to represent the most specific inducers of angiogenesis. Several members of both families have been shown to be constitutively expressed by AML blasts.17,20,22,23,31 Therefore, we measured mRNA levels of VEGF-A, VEGF-C, Ang1, Ang2, and Tie2 by qPCR in pretreatment samples from 90 patients younger than 61 years of age with de novo AML entered onto the AML SHG Hannover 95 trial. Although RFS of the entire group was slightly better than those of analyzed patients, no significant difference between analysis and nonanalysis groups could be found. One limitation of the study is that no plasma samples of the study patients were available; thus, analysis was restricted to investigation of mRNA expression of angiogenic factors. Therefore, exact data on protein concentrations in blood are unknown. However, given that no post-transcriptional regulation of these factors has been described, concerted mRNA and protein expression of these factors can be supposed. Furthermore, mRNA and protein expression of Ang2 by qPCR and ELISA from five human leukemic cell lines was determined. A positive correlation was found, supporting the validity of the use of cDNA for our investigation. Multivariate analysis revealed that expression of Ang2 and a good response on a day-15 postinduction chemotherapy bone marrow sample were the only independent predictors of OS in our patient cohort. The prognostic impact of persistent leukemia after induction chemotherapy has been confirmed by several groups.35–37 Because bad response to induction chemotherapy is correlated to adverse karyotypes, both groups of patients overlap to a high degree. Therefore, it was not possible to detect an independent prognostic value of karyotype in our cohort. Because of the small number of patients with specific chromosome aberrations, patients were grouped together into only two prognostic cohorts. It cannot be excluded by our analysis that individual translocations may result in a specific risk profile. The role of Ang2 in angiogenesis is still controversial. In initial studies, Ang2 has been shown to block the effect of Ang1 on endothelial cells in vitro. Furthermore, Ang2 competes with Ang1 for binding to their common receptor Tie2, and acts as a competitive inhibitor of Ang1. Transgenic overexpression of Ang2 resulted in insufficient vascular remodeling, as in Ang1 or Tie2 knockout mice, indicating that Ang2 could act as an antagonist of Ang1 in vivo.13 Furthermore, Lin et al38 demonstrated that neutralization of the effects of Ang1 by soluble Tie2 blocked angiogenesis and tumor growth in a mouse model. Recently, the role of Ang2 as a pure inhibitor of angiogenesis has been challenged. Emphasis has now been put on the concerted action of Ang2 and VEGF-A on endothelial cells. In several experimental models, Ang2 (in the absence of VEGF-A) led to endothelial cell apoptosis and vessel regression. In the presence of VEGF-A, Ang2 promoted endothelial cell proliferation and migration, thereby acting as a proangiogenic agent.14–16,39–41 The dependence of the effects of Ang2 by VEGF-A is also suggested by our results. Although Ang2 expression was an independent predictor of OS for the entire cohort, subgroup analysis indicated that the prognosis of patients with VEGF-A (and especially VEGF-C and Ang1) expression below the median was most dependent on the level of Ang2 production. In particular, patients with high Ang2 and low VEGF-C expression had an excellent long-term prognosis. On the other hand, in patients with high VEGF-C levels, prognosis was much less influenced by Ang2. Unfortunately, in our study no corresponding bone marrow biopsies were available; therefore, we could not directly correlate bone marrow angiogenesis with expression of VEGF-A, VEGF-C, and Ang2. Recently, several smaller studies have been published in which Ang2 expression was found in a variety of solid tumors.32,42,43 Ang2 could be detected in tumor cells and endothelial cells of invading tumor vessels in subgroups of patients. In the largest of these studies, expression of VEGF-A, Ang1, and Ang2 was analyzed in 236 patients with localized non-small-cell lung cancer. This investigation clearly showed that concerted expression of VEGF-A and Ang2 resulted in increased microvessel density and adverse long-term prognosis, supporting the model of cooperative effects of both types of growth factors.43 On the basis of the concept of cooperation between both classes of endothelial-specific growth factors, additional clinical and experimental research should be done to gain further insights into the dependence of neoangiogenesis on the interaction of Ang2 and VEGF-A or VEGF-C expression. If substantiated, therapeutic strategies directed against the action of VEGF-A and VEGF-C with simultaneous administration of Ang2 may be explored in experimental models to investigate enhanced antiangiogenic and antileukemic efficacy.
The authors indicated no potential conflicts of interest.
We thank the members of the AML SHG Hannover study group for providing samples.
Supported by a grant from Werner-Otto Stiftung Hamburg, Germany (S.L.), and a grant from Eppendorfer: Leukämiehilfe Hamburg and Roggenbuck Stiftung, Hamburg, Germany (U.M.G.). Presented in part at the annual meeting of the American Society of Hematology in Philadelphia, PA, December 6-10, 2002. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
was computed. 
