Leukemia Research 38 (2014) 662–665
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Interferon decreases VEGF levels in patients with chronic myeloid leukemia treated with imatinib夽 L. Legros a,b,∗ , J. Guilhot c , S. Huault b , F.X. Mahon d , C. Preudhomme e , F. Guilhot c , A.O. Hueber b , from the French CML Group (FI-LMC) a
Service d’Hématologie Clinique, Hôpital Archet 1, Nice, France Institut de Biologie Valrose (iBV), UMR CNRS 7277-UMR INSERM 1091, Nice, France c Inserm CIC 0802, CHU de Poitiers, France d Laboratoire d’Hématologie et Service des Maladies du Sang, Centre Hospitalier Universitaire de Bordeaux Université Bordeaux Ségalen, INSERM 1035, institut Bergonié, Bordeaux, France e Laboratoire d’hématologie, CHU de Lille, and Inserm U837, Lille, France b
a r t i c l e
i n f o
Article history: Received 26 October 2013 Received in revised form 24 December 2013 Accepted 19 January 2014 Available online 1 February 2014 Keywords: Vascular Endothelial Growth Factor (VEGF) Imatinib Chronic myeloid leukemia (CML) Angiogenesis Interferon-alpha
a b s t r a c t In chronic myeloid leukemia (CML), evidence is supporting the role of VEGF in growth, and survival of leukemia cells. The evaluation of plasma VEGF levels in 403 CML patients randomized within SPIRIT study to received imatinib-400 mg versus imatinib + cytarabine versus imatinib + interferon (IFN) versus imatinib-600 mg demonstrated that VEGF is an independent factor of BCR–ABL burden. VEGF low levels at diagnosis were associated with a progression-free survival of 100% at 48 months. Under treatment, significant lowest levels were observed in imatinib + IFN arm. These results support the use of VEGF as a parameter to predict CML evolution and let us to speculate about antiangiogenic properties of IFN. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder from a primitive hematopoietic cell associated with a characteristic chromosomal translocation called the Philadelphia chromosome and originates a chimeric BCR/ABL protein [1]. Maintenance of leukemic stem cells is supported by both the osteoblastic and vascular niche [2]. Endothelial cells in this vascular niche are capable of secreting several soluble cytokines such as Vascular Endothelial Growth Factor (VEGF) and VEGF is one of the most potent and specific regulators of angiogenesis by regulating several of endothelial cells’ functions, including mitogenesis, permeability and migration [3] but also by promoting leukemia cell proliferation and survival [4]. There is accumulating evidence that angiogenesis
夽 Programme Hospitalier de Recherche Clinique 2004 and 2006 from the French Minister of Health. ∗ Corresponding author at: Service d’Hématologie Clinique, Hôpital Archet 1, 151, Route de Saint Antoine de Ginestière, BP 3079, 06202 Nice Cedex 2, France. Tel.: +33 04 92 03 58 41; fax: +33 04 92 03 58 95. E-mail address: [email protected] (L. Legros). 0145-2126/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2014.01.010
is implicated in the outgrowth of BCR/ABL+ progenitors and CML progression [5–7]. The therapeutic strategy in CML has been totally modified with the development of imatinib, a specific inhibitor of BCR–ABL tyrosine kinase activity. Previously, we have studied the effect of imatinib on VEGF [8], which is elevated in CML at diagnosis [5]. Imatinib inhibits VEGF gene transcription by targeting the Sp1 and Sp3 transcription factors. In order to evaluate the impact of VEGF in the monitoring of CML patients treated with imatinib, we thus analyzed VEGF plasma levels in patients treated within the vast phase III prospective SPIRIT trial, a randomized comparison of imatinib 400 mg daily (IM 400) versus imatinib 600 mg daily (IM 600) versus IM400 in combination with Pegylated-interferon-␣2a (IFN) or with Cytarabine (Ara-C) in newly diagnosed chronic phase CML patients. 2. Patients, materials and methods 2.1. Patients Plasma was obtained from CML patients enrolled in the French trial SPIRIT, a phase III, multicenter, open-label, prospective trial,
L. Legros et al. / Leukemia Research 38 (2014) 662–665
663
upon written informed consent in accordance with the declaration of Helsinki (Clinical trial.gov: NCT00219739) [9]. Patients were eligible for the study if they were more than 18 years of age and had received a diagnosis of chronic phase, Philadelphia-positive or Philadelphia-negative and BCR–ABL positive CML within 3 months before study entry. Patients were randomized between the 3 experimental arms: IM 400 in combination with Pegylated-IFN or with Ara-C or IM 600. The reference arm was IM 400.
3. VEGF ELISA Samples were collected before starting the allocated treatment and every 3 months until 2 years in parallel to samples for molecular biology. Blood samples were centralized in the Laboratoire d’hématologie, and INSERM U837, CHU Lille, France and plasmas were collected following centrifugation (at 4000 rpm, 10 min) and frozen immediately at −20 ◦ C until analysis. Plasma samples were tested for VEGF-A concentrations using the sandwich enzyme-linked immunosorbent assay (ELISA) (Quantikine Human VEGF-A, R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. Each sample was tested in duplicate. VEGF levels were expressed as picogram per milliliter (pg/ml). The sensitivity of the kit is 5 pg/ml.
3.1. Responses assessment Complete haematological response (CHR), complete cytogenetic response (CCyR) and major molecular rates (MMR) are defined according ELN criteria [10]. Molecular monitoring has been described elsewhere [9].
3.2. Statistical analysis The values of plasma VEGF were described as median, interquartile ranges (low quartile–high quartile [LQ–HQ]). Data analyses were performed by using SAS software (SAS, Cary, NC, USA). In comparison of plasma levels distribution between groups, statistical significance was determined with the Wilcoxon Rank sum test or the Kruskall–Wallis test, as appropriate. For paired analyses Wilcoxon sign rank sum test and Spearman’s correlation test were used. p-Values < 0.05 were considered statistically significant. VEGF level was analyzed according to age, gender, Sokal index, molecular response, platelets, additional chromosomal abnormalities, blood and marrow blast percentage. Progression free survival was estimated by the Kaplan Meier method and compared within groups with the log-rank test.
4. Results 4.1. Patients’ characteristics The 403 first patients enrolled in the SPIRIT trial were analyzed. Baseline demographics and disease characteristics before randomization are summarized in Table 1. The sex ratio (males/females) was 1.6. The median age at diagnosis was 52 years (range, 19–82 years). Sokal scores were low (38%), intermediate (38%), and high (24%). The median follow up was 56 months (3–81 months). After randomization, 101 patients received IM 400, 99 patients IM 600, 102 patients IM 400 + Ara-C and 101 patients IM 400 + IFN. Overall at 3 months 73% of patients achieved CHR. The CCyR rate at 12 months was 64%; MMR rate at 12 and 24 months were 48%, and 56% respectively.
Fig. 1. (A) Box plots of VEGF plasma level at baseline (M0) and during treatment. VEGF plasma level was evaluated at baseline (M0) in 403 patients, at 3 months (M3) in 309 patients, at 6 months (M6) in 261 patients, at 9 months (M9) in 197 patients and at 12 months (M12) in 178 patients. (B) Kaplan–Meier estimates progressionfree survival. Progression was defined by any of the following events, whichever came first: death, accelerated-phase or blast-crisis CML. (C) Box plots of VEGF plasma level at 12 months according arm treatment. VEGF plasma level was evaluated at 12 months (M12) according arm treatment: imatinib 400 mg daily (IM400), (IM) in combination with Pegylated-interferon-␣2a (IFN) or with Cytarabine (Ara-C) or imatinib 600 mg daily (IM 600).
4.2. VEGF plasma levels before treatment At baseline, median plasma VEGF level was 886.5 pg/ml [LQ–HQ: 327.5–1711] (Fig. 1A). Baseline VEGF levels according to patient’s characteristics are described in Table 1. We next analyzed correlation between VEGF levels and usual prognosis factors such age, Sokal score, and additional chromosomal abnormalities (ACA). VEGF levels were only correlated to Sokal risk score and to platelets count (Table 1). Given the ability of VEGF to facilitate leukemic growth and progression of AML in paracrine [11] or autocrine pathways [6,12], we
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L. Legros et al. / Leukemia Research 38 (2014) 662–665
Table 1 Baseline characteristics of patients. Baseline characteristics
n (%)
Median of VEGF pg/ml (range)
p value
Sex Female Male
155 (38) 248 (62)
1106.4 (41.4–4054.0) 826.4 (19.3–3179.0)
p = 0.052
Sokal index Low Intermediate High
153 (38) 155 (38) 95 (24)
706.0 (34.7–2735.5) 891.3 (41.4–3179.0) 1295.4 (19.3–4054.0)
p < 0.01
Platelets 1500 × 1012 /L
378 (94) 19 (5) 6 (2)
866.7 (19.3–3179.0) 1057.6 (82.2–4054.0) 2239.0 (541.4–2434.8)
p = 0.045
Blood blasts Absence Presence NA
195 (48) 192 (48) 16 (4)
941.2 (20.3–3179.0) 952.2 (19.3–4054.0) –
p = 0.96
Marrow blasts Absence Presence NA
208 (52) 160 (40) 35 (9)
1035.0 (20.3–3179.0) 826.5 (19.3–4054.0) –
p = 0.28
339
891.3 (41.4–4053.9)
20 17 27
414.3 (20.2–2573.5) 541.3 (19.3–2511.7) 1295.4 (34.7–2691.5)
Karyotype No additional abnormality With additional abnormalities Del Y Poor prognosis Unclassifiable
p = 0.20
Abbreviation: NA: not applicable.
investigated the correlation between VEGF and blast percentage regardless of blood or bone marrow origin (Table 1) but we did not find any correlation. To refine our analysis in aim to evaluate the prognostic value of VEGF on treatment response, VEGF plasma levels at baseline were distributed in 3 groups according to quartiles: low (Q < 25) (n = 100), intermediate (Q25–75) (n = 202) and high (Q > 75) (n = 101). We failed to find a significant correlation between baseline VEGF levels and cytogenetic or molecular response to therapy (data not shown).
4.3. VEGF plasma level outcome following treatment We observed a significant decrease of VEGF levels during treatment when compared to VEGF amounts at baseline (p < 0.05) (Fig. 1A). At 3, 6, 9 and 12 months of imatinib, median plasma VEGF level was 100.5 pg/ml [LQ–HQ: 54.0–180.3 pg/ml], 90.1 pg/ml [LQ–HQ: 54.0–167.0], 79.9 pg/ml [LQ–HQ: 49.8–140.1], and 84.3 pg/ml [LQ–HQ: 49.6–157.7]. This decay is independent from response to treatment. Indeed, we failed to found a significant correlation between VEGF levels under treatment and cytogenetic or molecular response to therapy (data not shown). As VEGF is not correlated to BCR–ABL burden, nor to treatment response, we evaluated its impact on CML evolution using the quartile repartition of VEGF level. Interestingly, the lower quartile of VEGF at diagnosis was associated with a 100% progression free survival (PFS) at 48 months (Fig. 1B). In the last part of our study we analyzed the VEGF level according to treatment arm. At baseline, VEGF levels were comparable within the 4 arms of treatment (data not shown). At 12 months of treatment, the lowest levels of VEGF were observed in patients treated with IM in association with IFN (p = 0.02) in comparison with those treated with IM 400 alone (Fig. 1C). The median plasma VEGF level in IM 400 arm, IM 600 arm, IM-IFN arm, and IM-Ara-C arm were respectively 99.4 pg/ml [LQ–HQ: 61.2–192.5], 96.2 pg/ml [LQ–HQ: 56.9–151.0], 62.3 pg/ml [LQ–HQ: 45.4–115.9], and 94.3 pg/ml [LQ–HQ: 44.3–199.8] (Fig. 1C).
5. Discussion In aim to evaluate the impact of VEGF in the monitoring of CML patients treated with imatinib, we have analyzed VEGF plasma levels in the first 403 patients treated within the SPIRIT study. Sex ratio, median age, Sokal risk distribution and cytogenetic and molecular response rates of this cohort were similar to that observed in the overall SPIRIT study [9]. We found, at diagnosis, an increased level of VEGF around 8 fold confirming previous studies [5,8,13,14]. For the record we found previously that the median VEGF was 112.1 pg/ml (range: 54.4–177.2) in healthy volunteers’ plasma [8]. At baseline, VEGF levels were correlated to platelets count partially explaining the correlation observed with Sokal index since platelets count is one of factors used for Sokal index calculation. During the first 3 months of treatment, VEGF levels decreased to reach normal levels and remained stable thereafter at a low level (Fig. 1A). Our results are in accordance with our previous study, which showed that imatinib decreases VEGF levels [8]. However, we failed to find a significant correlation between baseline VEGF levels or VEGF levels under treatment and cytogenetic or molecular response to therapy. Finally, we can conclude that VEGF level and BCR-ABL burden are two independent factors. We showed that lower VEGF levels at diagnosis were associated with a 100% of PFS at 48 months (Fig. 1B). There is increasing interest in developing strategies to identify, as early as possible, patients who will not respond optimally to imatinib so that they can be offered an alternative tyrosine kinase inhibitor. Recently early assessment of molecular response has been proposed to identify patients destined to fare poorly, thereby allowing early clinical intervention [15,16]. VEGF evaluation could be could be a useful parameter available at diagnosis. We have also shown that the lowest levels of VEGF were observed in patients treated with IM in association with IFN. Such lower levels of VEGF and the absence of correlation between VEGF levels and molecular response led us to postulate that the effect of IFN in CML could be in part mediated by a direct effect on VEGF
L. Legros et al. / Leukemia Research 38 (2014) 662–665
production. Moreover, we have already observed a decrease of VEGF levels in IM-resistant patients treated with IFN alone [14]. This led us to hypothesize that IFN could potentiate antiangiogenic activity of imatinib. A previous study demonstrating that IFN is able to inhibit VEGF gene transcription corroborates our allegation [17]. Recently, several studies have demonstrated that the addition of IFN to imatinib results in significantly higher molecular responses rates in patients [9,18] and thus potentially promoting the revival of IFN in CML [19]. Precise mechanisms of IFN action in CML are not completely elucidated. The hematopoietic stem cell (HSC) niche is a rich and complex environment [20,21] and IFN could impact not only on leukemic HSC but also in an antiangiogenic way. Conflict of interest statement There is no relevant conflicts of interest disclosure regarding this study. Acknowledgement We thank Pr. Pierre-Simon Rohrlich CHU of Nice, and Dr. Franck Nicolini, hospices civils de Lyon, for critically reading of the manuscript. Authorship contributions: L.L. designed the study, was the principal investigator, performed data analysis and wrote the paper. J.G. performed statistical analysis. L.L. and S.H. performed the laboratory work. C.P. and F.X.M. performed the molecular analysis, reviewed and approved the final version of the report. A.O.H. reviewed and approved the final version of the report. F.G. designed the SPIRIT study, critically reviewed the manuscript, and approved it in its final version. References [1] Sawyers CL. Chronic myeloid leukemia. N Engl J Med 1999;340:1330–40. [2] Konopleva M, Tabe Y, Zeng Z, Andreeff M. Therapeutic targeting of microenvironmental interactions in leukemia: mechanisms and approaches. Drug Resist Update 2009;12:103–13. [3] Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin Oncol 2002;29:10–4. [4] Hatfield K, Ryningen A, Corbascio M, Bruserud O. Microvascular endothelial cells increase proliferation and inhibit apoptosis of native human acute myelogenous leukemia blasts. Int J Cancer 2006;119:2313–21. [5] Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, Thomas D, et al. Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes. Blood 2000;96:2240–5.
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[6] Dias S, Hattori K, Zhu Z, Heissig B, Choy M, Lane W, et al. Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J Clin Invest 2000;106:511–21. [7] Korkolopoulou P, Viniou N, Kavantzas N, Patsouris E, Thymara I, Pavlopoulos PM, et al. Clinicopathologic correlations of bone marrow angiogenesis in chronic myeloid leukemia: a morphometric study. Leukemia 2003;17: 89–97. [8] Legros L, Bourcier C, Jacquel A, Mahon FX, Cassuto JP, Auberger P, et al. Imatinib mesylate (STI571) decreases the vascular endothelial growth factor plasma concentration in patients with chronic myeloid leukemia. Blood 2004;104:495–501. [9] Preudhomme C, Guilhot J, Nicolini FE, Guerci-Bresler A, Rigal-Huguet F, Maloisel F, et al. Imatinib plus peginterferon alfa-2a in chronic myeloid leukemia. N Engl J Med 2010;363:2511–21. [10] Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J, et al. Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol 2009;27:6041–51. [11] Fiedler W, Graeven U, Ergun S, Verago S, Kilic N, Stockschlader M, et al. Vascular endothelial growth factor, a possible paracrine growth factor in human acute myeloid leukemia. Blood 1997;89:1870–5. [12] Bellamy W, Richter L, Sirjani D, Roxas C, Glinsmann-Gibson B, Frutiger Y, et al. Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes. Blood 2001;97:1427–34. [13] Verstovsek S, Kantarjian H, Manshouri T, Cortes J, Giles FJ, Rogers A, et al. Prognostic significance of cellular vascular endothelial growth factor expression in chronic phase chronic myeloid leukemia. Blood 2002;99:2265–7. [14] Nicolini FE, Hayette S, Legros L, Rousselot P, Maloisel F, Tulliez M, et al. Pegylated IFN-alpha2a combined to imatinib mesylate 600 mg daily can induce complete cytogenetic and molecular responses in a subset of chronic phase CML patients refractory to IFN alone or to imatinib 600 mg daily alone. Leuk Res 2011;35:80–6. [15] Marin DF, Ibrahim AR, Ibrahim AF, Lucas C, Lucas CF, Gerrard G, et al. Clark RE assessment of BCR–ABL1 transcript levels at 3 months is the only requirement for predicting outcome for patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. J Clin Oncol 2012;30:232–8. [16] Ohm LF, Arvidsson I, Arvidsson IF, Barbany G, Barbany GF, Hast R, et al. Early landmark analysis of imatinib treatment in CML chronic phase: less than 10% BCR–ABL by FISH at 3 months associated with improved long-term clinical outcome. Am J Hematol. Am J Hematol 2012;87:760–5. [17] von Marschall Z, Scholz A, Cramer T, Schafer G, Schirner M, Oberg K, et al. Effects of interferon alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis. J Natl Cancer Inst 2003;95: 437–48. [18] Simonsson B, Gedde-Dahl T, Markevarn B, Remes K, Stentoft J, Almqvist A, et al. Combination of pegylated IFN-alpha2b with imatinib increases molecular response rates in patients with low- or intermediate-risk chronic myeloid leukemia. Blood 2012;118:3228–35. [19] Talpaz M, Hehlmann R, Quintas-Cardama A, Mercer J, Cortes J. Re-emergence of interferon-alpha in the treatment of chronic myeloid leukemia. Leukemia 2012;27:803–12. [20] Hsu YC, Fuchs E. A family business: stem cell progeny join the niche to regulate homeostasis. Nat Rev Mol Cell Biol 2012;13:103–14. [21] Wang LD, Wagers AJ. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nat Rev Mol Cell Biol 2011;12:643–55.
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Interferon decreases VEGF levels in patients with chronic myeloid leukemia treated with imatinib夽 L. Legros a,b,∗ , J. Guilhot c , S. Huault b , F.X. Mahon d , C. Preudhomme e , F. Guilhot c , A.O. Hueber b , from the French CML Group (FI-LMC) a
Service d’Hématologie Clinique, Hôpital Archet 1, Nice, France Institut de Biologie Valrose (iBV), UMR CNRS 7277-UMR INSERM 1091, Nice, France c Inserm CIC 0802, CHU de Poitiers, France d Laboratoire d’Hématologie et Service des Maladies du Sang, Centre Hospitalier Universitaire de Bordeaux Université Bordeaux Ségalen, INSERM 1035, institut Bergonié, Bordeaux, France e Laboratoire d’hématologie, CHU de Lille, and Inserm U837, Lille, France b
a r t i c l e
i n f o
Article history: Received 26 October 2013 Received in revised form 24 December 2013 Accepted 19 January 2014 Available online 1 February 2014 Keywords: Vascular Endothelial Growth Factor (VEGF) Imatinib Chronic myeloid leukemia (CML) Angiogenesis Interferon-alpha
a b s t r a c t In chronic myeloid leukemia (CML), evidence is supporting the role of VEGF in growth, and survival of leukemia cells. The evaluation of plasma VEGF levels in 403 CML patients randomized within SPIRIT study to received imatinib-400 mg versus imatinib + cytarabine versus imatinib + interferon (IFN) versus imatinib-600 mg demonstrated that VEGF is an independent factor of BCR–ABL burden. VEGF low levels at diagnosis were associated with a progression-free survival of 100% at 48 months. Under treatment, significant lowest levels were observed in imatinib + IFN arm. These results support the use of VEGF as a parameter to predict CML evolution and let us to speculate about antiangiogenic properties of IFN. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder from a primitive hematopoietic cell associated with a characteristic chromosomal translocation called the Philadelphia chromosome and originates a chimeric BCR/ABL protein [1]. Maintenance of leukemic stem cells is supported by both the osteoblastic and vascular niche [2]. Endothelial cells in this vascular niche are capable of secreting several soluble cytokines such as Vascular Endothelial Growth Factor (VEGF) and VEGF is one of the most potent and specific regulators of angiogenesis by regulating several of endothelial cells’ functions, including mitogenesis, permeability and migration [3] but also by promoting leukemia cell proliferation and survival [4]. There is accumulating evidence that angiogenesis
夽 Programme Hospitalier de Recherche Clinique 2004 and 2006 from the French Minister of Health. ∗ Corresponding author at: Service d’Hématologie Clinique, Hôpital Archet 1, 151, Route de Saint Antoine de Ginestière, BP 3079, 06202 Nice Cedex 2, France. Tel.: +33 04 92 03 58 41; fax: +33 04 92 03 58 95. E-mail address: [email protected] (L. Legros). 0145-2126/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2014.01.010
is implicated in the outgrowth of BCR/ABL+ progenitors and CML progression [5–7]. The therapeutic strategy in CML has been totally modified with the development of imatinib, a specific inhibitor of BCR–ABL tyrosine kinase activity. Previously, we have studied the effect of imatinib on VEGF [8], which is elevated in CML at diagnosis [5]. Imatinib inhibits VEGF gene transcription by targeting the Sp1 and Sp3 transcription factors. In order to evaluate the impact of VEGF in the monitoring of CML patients treated with imatinib, we thus analyzed VEGF plasma levels in patients treated within the vast phase III prospective SPIRIT trial, a randomized comparison of imatinib 400 mg daily (IM 400) versus imatinib 600 mg daily (IM 600) versus IM400 in combination with Pegylated-interferon-␣2a (IFN) or with Cytarabine (Ara-C) in newly diagnosed chronic phase CML patients. 2. Patients, materials and methods 2.1. Patients Plasma was obtained from CML patients enrolled in the French trial SPIRIT, a phase III, multicenter, open-label, prospective trial,
L. Legros et al. / Leukemia Research 38 (2014) 662–665
663
upon written informed consent in accordance with the declaration of Helsinki (Clinical trial.gov: NCT00219739) [9]. Patients were eligible for the study if they were more than 18 years of age and had received a diagnosis of chronic phase, Philadelphia-positive or Philadelphia-negative and BCR–ABL positive CML within 3 months before study entry. Patients were randomized between the 3 experimental arms: IM 400 in combination with Pegylated-IFN or with Ara-C or IM 600. The reference arm was IM 400.
3. VEGF ELISA Samples were collected before starting the allocated treatment and every 3 months until 2 years in parallel to samples for molecular biology. Blood samples were centralized in the Laboratoire d’hématologie, and INSERM U837, CHU Lille, France and plasmas were collected following centrifugation (at 4000 rpm, 10 min) and frozen immediately at −20 ◦ C until analysis. Plasma samples were tested for VEGF-A concentrations using the sandwich enzyme-linked immunosorbent assay (ELISA) (Quantikine Human VEGF-A, R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. Each sample was tested in duplicate. VEGF levels were expressed as picogram per milliliter (pg/ml). The sensitivity of the kit is 5 pg/ml.
3.1. Responses assessment Complete haematological response (CHR), complete cytogenetic response (CCyR) and major molecular rates (MMR) are defined according ELN criteria [10]. Molecular monitoring has been described elsewhere [9].
3.2. Statistical analysis The values of plasma VEGF were described as median, interquartile ranges (low quartile–high quartile [LQ–HQ]). Data analyses were performed by using SAS software (SAS, Cary, NC, USA). In comparison of plasma levels distribution between groups, statistical significance was determined with the Wilcoxon Rank sum test or the Kruskall–Wallis test, as appropriate. For paired analyses Wilcoxon sign rank sum test and Spearman’s correlation test were used. p-Values < 0.05 were considered statistically significant. VEGF level was analyzed according to age, gender, Sokal index, molecular response, platelets, additional chromosomal abnormalities, blood and marrow blast percentage. Progression free survival was estimated by the Kaplan Meier method and compared within groups with the log-rank test.
4. Results 4.1. Patients’ characteristics The 403 first patients enrolled in the SPIRIT trial were analyzed. Baseline demographics and disease characteristics before randomization are summarized in Table 1. The sex ratio (males/females) was 1.6. The median age at diagnosis was 52 years (range, 19–82 years). Sokal scores were low (38%), intermediate (38%), and high (24%). The median follow up was 56 months (3–81 months). After randomization, 101 patients received IM 400, 99 patients IM 600, 102 patients IM 400 + Ara-C and 101 patients IM 400 + IFN. Overall at 3 months 73% of patients achieved CHR. The CCyR rate at 12 months was 64%; MMR rate at 12 and 24 months were 48%, and 56% respectively.
Fig. 1. (A) Box plots of VEGF plasma level at baseline (M0) and during treatment. VEGF plasma level was evaluated at baseline (M0) in 403 patients, at 3 months (M3) in 309 patients, at 6 months (M6) in 261 patients, at 9 months (M9) in 197 patients and at 12 months (M12) in 178 patients. (B) Kaplan–Meier estimates progressionfree survival. Progression was defined by any of the following events, whichever came first: death, accelerated-phase or blast-crisis CML. (C) Box plots of VEGF plasma level at 12 months according arm treatment. VEGF plasma level was evaluated at 12 months (M12) according arm treatment: imatinib 400 mg daily (IM400), (IM) in combination with Pegylated-interferon-␣2a (IFN) or with Cytarabine (Ara-C) or imatinib 600 mg daily (IM 600).
4.2. VEGF plasma levels before treatment At baseline, median plasma VEGF level was 886.5 pg/ml [LQ–HQ: 327.5–1711] (Fig. 1A). Baseline VEGF levels according to patient’s characteristics are described in Table 1. We next analyzed correlation between VEGF levels and usual prognosis factors such age, Sokal score, and additional chromosomal abnormalities (ACA). VEGF levels were only correlated to Sokal risk score and to platelets count (Table 1). Given the ability of VEGF to facilitate leukemic growth and progression of AML in paracrine [11] or autocrine pathways [6,12], we
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L. Legros et al. / Leukemia Research 38 (2014) 662–665
Table 1 Baseline characteristics of patients. Baseline characteristics
n (%)
Median of VEGF pg/ml (range)
p value
Sex Female Male
155 (38) 248 (62)
1106.4 (41.4–4054.0) 826.4 (19.3–3179.0)
p = 0.052
Sokal index Low Intermediate High
153 (38) 155 (38) 95 (24)
706.0 (34.7–2735.5) 891.3 (41.4–3179.0) 1295.4 (19.3–4054.0)
p < 0.01
Platelets 1500 × 1012 /L
378 (94) 19 (5) 6 (2)
866.7 (19.3–3179.0) 1057.6 (82.2–4054.0) 2239.0 (541.4–2434.8)
p = 0.045
Blood blasts Absence Presence NA
195 (48) 192 (48) 16 (4)
941.2 (20.3–3179.0) 952.2 (19.3–4054.0) –
p = 0.96
Marrow blasts Absence Presence NA
208 (52) 160 (40) 35 (9)
1035.0 (20.3–3179.0) 826.5 (19.3–4054.0) –
p = 0.28
339
891.3 (41.4–4053.9)
20 17 27
414.3 (20.2–2573.5) 541.3 (19.3–2511.7) 1295.4 (34.7–2691.5)
Karyotype No additional abnormality With additional abnormalities Del Y Poor prognosis Unclassifiable
p = 0.20
Abbreviation: NA: not applicable.
investigated the correlation between VEGF and blast percentage regardless of blood or bone marrow origin (Table 1) but we did not find any correlation. To refine our analysis in aim to evaluate the prognostic value of VEGF on treatment response, VEGF plasma levels at baseline were distributed in 3 groups according to quartiles: low (Q < 25) (n = 100), intermediate (Q25–75) (n = 202) and high (Q > 75) (n = 101). We failed to find a significant correlation between baseline VEGF levels and cytogenetic or molecular response to therapy (data not shown).
4.3. VEGF plasma level outcome following treatment We observed a significant decrease of VEGF levels during treatment when compared to VEGF amounts at baseline (p < 0.05) (Fig. 1A). At 3, 6, 9 and 12 months of imatinib, median plasma VEGF level was 100.5 pg/ml [LQ–HQ: 54.0–180.3 pg/ml], 90.1 pg/ml [LQ–HQ: 54.0–167.0], 79.9 pg/ml [LQ–HQ: 49.8–140.1], and 84.3 pg/ml [LQ–HQ: 49.6–157.7]. This decay is independent from response to treatment. Indeed, we failed to found a significant correlation between VEGF levels under treatment and cytogenetic or molecular response to therapy (data not shown). As VEGF is not correlated to BCR–ABL burden, nor to treatment response, we evaluated its impact on CML evolution using the quartile repartition of VEGF level. Interestingly, the lower quartile of VEGF at diagnosis was associated with a 100% progression free survival (PFS) at 48 months (Fig. 1B). In the last part of our study we analyzed the VEGF level according to treatment arm. At baseline, VEGF levels were comparable within the 4 arms of treatment (data not shown). At 12 months of treatment, the lowest levels of VEGF were observed in patients treated with IM in association with IFN (p = 0.02) in comparison with those treated with IM 400 alone (Fig. 1C). The median plasma VEGF level in IM 400 arm, IM 600 arm, IM-IFN arm, and IM-Ara-C arm were respectively 99.4 pg/ml [LQ–HQ: 61.2–192.5], 96.2 pg/ml [LQ–HQ: 56.9–151.0], 62.3 pg/ml [LQ–HQ: 45.4–115.9], and 94.3 pg/ml [LQ–HQ: 44.3–199.8] (Fig. 1C).
5. Discussion In aim to evaluate the impact of VEGF in the monitoring of CML patients treated with imatinib, we have analyzed VEGF plasma levels in the first 403 patients treated within the SPIRIT study. Sex ratio, median age, Sokal risk distribution and cytogenetic and molecular response rates of this cohort were similar to that observed in the overall SPIRIT study [9]. We found, at diagnosis, an increased level of VEGF around 8 fold confirming previous studies [5,8,13,14]. For the record we found previously that the median VEGF was 112.1 pg/ml (range: 54.4–177.2) in healthy volunteers’ plasma [8]. At baseline, VEGF levels were correlated to platelets count partially explaining the correlation observed with Sokal index since platelets count is one of factors used for Sokal index calculation. During the first 3 months of treatment, VEGF levels decreased to reach normal levels and remained stable thereafter at a low level (Fig. 1A). Our results are in accordance with our previous study, which showed that imatinib decreases VEGF levels [8]. However, we failed to find a significant correlation between baseline VEGF levels or VEGF levels under treatment and cytogenetic or molecular response to therapy. Finally, we can conclude that VEGF level and BCR-ABL burden are two independent factors. We showed that lower VEGF levels at diagnosis were associated with a 100% of PFS at 48 months (Fig. 1B). There is increasing interest in developing strategies to identify, as early as possible, patients who will not respond optimally to imatinib so that they can be offered an alternative tyrosine kinase inhibitor. Recently early assessment of molecular response has been proposed to identify patients destined to fare poorly, thereby allowing early clinical intervention [15,16]. VEGF evaluation could be could be a useful parameter available at diagnosis. We have also shown that the lowest levels of VEGF were observed in patients treated with IM in association with IFN. Such lower levels of VEGF and the absence of correlation between VEGF levels and molecular response led us to postulate that the effect of IFN in CML could be in part mediated by a direct effect on VEGF
L. Legros et al. / Leukemia Research 38 (2014) 662–665
production. Moreover, we have already observed a decrease of VEGF levels in IM-resistant patients treated with IFN alone [14]. This led us to hypothesize that IFN could potentiate antiangiogenic activity of imatinib. A previous study demonstrating that IFN is able to inhibit VEGF gene transcription corroborates our allegation [17]. Recently, several studies have demonstrated that the addition of IFN to imatinib results in significantly higher molecular responses rates in patients [9,18] and thus potentially promoting the revival of IFN in CML [19]. Precise mechanisms of IFN action in CML are not completely elucidated. The hematopoietic stem cell (HSC) niche is a rich and complex environment [20,21] and IFN could impact not only on leukemic HSC but also in an antiangiogenic way. Conflict of interest statement There is no relevant conflicts of interest disclosure regarding this study. Acknowledgement We thank Pr. Pierre-Simon Rohrlich CHU of Nice, and Dr. Franck Nicolini, hospices civils de Lyon, for critically reading of the manuscript. Authorship contributions: L.L. designed the study, was the principal investigator, performed data analysis and wrote the paper. J.G. performed statistical analysis. L.L. and S.H. performed the laboratory work. C.P. and F.X.M. performed the molecular analysis, reviewed and approved the final version of the report. A.O.H. reviewed and approved the final version of the report. F.G. designed the SPIRIT study, critically reviewed the manuscript, and approved it in its final version. References [1] Sawyers CL. Chronic myeloid leukemia. N Engl J Med 1999;340:1330–40. [2] Konopleva M, Tabe Y, Zeng Z, Andreeff M. Therapeutic targeting of microenvironmental interactions in leukemia: mechanisms and approaches. Drug Resist Update 2009;12:103–13. [3] Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin Oncol 2002;29:10–4. [4] Hatfield K, Ryningen A, Corbascio M, Bruserud O. Microvascular endothelial cells increase proliferation and inhibit apoptosis of native human acute myelogenous leukemia blasts. Int J Cancer 2006;119:2313–21. [5] Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, Thomas D, et al. Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes. Blood 2000;96:2240–5.
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