Bevacizumab reduces VEGF expression in patients with ... - Nature

8 downloads 233 Views 120KB Size Report
Mar 1, 2007 - kinase inhibitor and downstream signal pathway inhibitor such as rapamycin, as ... receptor tyrosine kinase genes. ... After 2 days, cell proliferation was measured by thymidine uptake. ... coagulation and clinical chemistry).
Letters to the Editor

1310 kinase inhibitor and downstream signal pathway inhibitor such as rapamycin, as well as AZD6244, may be a potential treatment strategy. In summary, ZD6474 may be an effective treatment for individuals with AML that possess mutations in receptor tyrosine kinase genes. In addition, the combined administration of ZD6474 and AZD6244 to patients with AML warrants further investigation.

Acknowledgements This work was supported in part by Kanae Foundation for the Promotion of the Medical Science, Public Trust of Haraguchi Memorial Cancer Research Fund, AstraZeneca Research Grant and the Fund for Academic Research from Kochi University. HPK is supported by NIH grants, as well as the Inger Fund and the Parker Hughes Trust.

C Nishioka1, T Ikezoe1, A Takeshita1, J Yang1, T Tasaka2, Y Yang1,3, Y Kuwayama1, N Komatsu1, K Togitani1, HP Koeffler4 and H Taguchi1 1 Department of Hematology and Respiratory Medicine, Kochi University, Nankoku, Kochi, Japan; 2 Department of Clinical Pathology and Laboratory Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan; 3 Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing, China and

4 Department of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USA E-mail: [email protected]

References 1 Wedge SR, Ogilvie DJ, Dukes M, Kendrew J, Chester R, Jackson JA et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res 2002; 62: 4645–4655. 2 Yang Y, Ikezoe T, Nishioka C, Taguchi T, Koeffler HP, Taguchi H. ZD6474 induces growth arrest and apoptosis of GIST-T1 cells and this growth inhibition is enhanced by concomitant use of sunitinib. Cancer Sci 2006; 97: 1404–1409. 3 Staehler M, Rohrmann K, Haseke N, Stief CG, Siebels M. Targeted agents for the treatment of advanced renal cell carcinoma. Curr Drug Targets 2005; 6: 835–846. 4 Fiedler W, Serve H, Dohner H, Schwittay M, Ottmann OG, O’Farrell AM et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005; 105: 986–993. 5 Ikezoe T, Nishioka C, Tasaka T, Yang Y, Komatsu N, Togitani K et al. The anti-tumor effects of sunitinib (formerly SU11248) against a variety of human hematological malignancies: enhancement of growth inhibition via inhibition of mTOR signaling. Mol Cancer Ther 2006; 5: 2522–2530.

Figure 2 AZD6244 potentiates the action of ZD6474 in leukemia cells. (a), EOL-1 or MV4-11 cells were cultured with ZD6474 and/or AZD6244 (0.125–1 mM). After 2 days, cell proliferation was measured by thymidine uptake. The percent inhibition was graphed and the concentration of each compound that induced 50, 75 or 90% growth inhibition (IC50, IC75, IC90) was determined (data not shown). The combination index (CI) of ZD6474 and AZD6244 at various dose effects (IC50, IC75, IC90) was calculated using the median effect method. CI values less than 1 indicate synergy, a CI equal to 1 indicates an additive effect, and a CI greater than 1 indicates antagonism between the two agents. (b) Cell cycle analysis. EOL-1 cells were cultured with ZD6474 and/or AZD6244 (0.25 mM). After 2 days, the cell cycle distribution of these cells was analyzed. The statistical significance of difference between populations in pre-G1 or S phase of cell cycle induced by either ZD6474 or AZD6244 alone and those induced by a combination of both was determined by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison tests. Results represent the mean7s.d. of three experiments performed in triplicate; nPo0.01, with respect to either compound alone. (c) Annexin V staining. EOL-1 cells were cultured with ZD6474 and/or AZD6244 (0.25 or 0.5 mM). After 2 days, cells were stained with annexin V/propidium iodide and analyzed by a flow cytometry. The statistical significance of difference between annexin V positive populations induced by either ZD6474 or AZD6244 alone and those induced by combination of both was determined by ANOVA followed by Bonferroni’s multiple comparison tests. Results represent the mean7s.d. of three experiments performed in triplicate; nPo0.01, with respect to either compound alone. (d) FACScan analysis of p-ERK. EOL-1 cells were cultured with either ZD6474 (0.25–1 mM) or control diluent. After 24 h, cells were exposed to either AZD6244 (0.25 mM) or control diluent for 15 min. Cells were harvested, incubated with anti-p-ERK antibody for 30 min at room temperature, and analyzed by flow cytometry. Results represent one of the experiments performed twice in duplicate plate. p-ERK-positive population was quantified using the CellQuest software package. For figure see previous page.

Bevacizumab reduces VEGF expression in patients with relapsed and refractory acute myeloid leukemia without clinical antileukemic activity

Leukemia (2007) 21, 1310–1312. doi:10.1038/sj.leu.2404632; published online 1 March 2007

Angiogenesis is an absolute requirement for tumor growth and metastasis, and is an attractive target for biologically based cancer therapies. Vascular endothelial growth factor (VEGF, VEGF-A) is one of the most potent promoters of angiogenesis whose functions are mainly mediated through the tyrosine kinase receptor VEGFR-2 (also known as KDR or Flk-1). Comparable to solid tumors, there is growing evidence that the VEGF/VEGFR2 signaling pathway plays an important role Leukemia

in leukemic cell growth.1–4 Clinical trials with VEGF inhibition in a variety of malignancies are ongoing. The best-studied and most advanced approach to VEGF inhibition is the recombinant humanized monoclonal antibody bevacizumab (BV). We have treated patients with relapsed or refractory acute myeloid leukemia (AML), not qualifying for further intensive cytotoxic chemotherapy, in order to investigate single agent BV in AML. Nine patients were treated between May 2005 and May 2006. All patients signed informed consent before enrollment. A summary of baseline patient characteristics is shown in Table 1. Patients were between 46 and 81 years old (median:

Letters to the Editor

1311 63 years), not medically fit to endure further conventional chemotherapy. Refractory AML was defined as primary resistance against at least two induction therapy regimens or Table 1

Baseline patient characteristics

Characteristic

No.

Patients enrolled Sex Male Female

9

Age, years, median (range) ECOG performance status, median (range) De novo AML

63 (46–81) 1 (0–3) 8

FAB type M0 M1 M2 M4 eo M5 Biphenotypic

1 2 2 1 1 1

4 5

Secondary AML Prior CMML

1

Stage of disease First relapse Median CR 1 duration Refractory Primary refractory Multirefractorya

2 7 months (6–8) 7 3 4

Biologic features Secondary AML Adverse cytogeneticsb

1 7

No. of cycles received 1 2 3

9 5 2

Abbreviations: AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; CR, complete remission; ECOG, Eastern Cooperative Oncology group; FAB, French American British classification. a Refractory to 42 induction regimens or to reinduction after first relapse. b inv (3) (q21q26), +8, +16, complex/hyperdiploid karyotype, transl. chromosome 5 and 8.

Table 2

relapsed patients who were refractory against at least one reinduction protocol. Seven patients suffered from refractory AML (three primary induction failure, four refractory to X2 induction regimens, or to reinduction after first relapse). Two patients had AML in first relapse (median duration of first complete remission (CR) 7 months; range 7–9 months) of whom one patient had secondary AML evolved from chronic myelomonocytic leukemia. Cytogenetics was abnormal in seven patients, including trisomy 8 and complex cytogenetics (Table 1). All patients had received dose-intensive infusional ara-C and/or high-dose bolus ara-C in combination with anthracycline or mitoxantrone as induction and/or consolidation regimens. BV (Avastin, Roche Pharma) at a dose of 10 mg/kg body weight was administered as a single i.v. infusion over 60–90 min on days 1, 14 and 35 (cycle 1–3). Acetaminophen and antihistamines (clemastin, ranitidin) were administered before infusion to reduce the risk of allergic reactions. To assess the clinical and molecular response to VEGF inhibition with BV, bone marrow aspiration and biopsy were performed before the initiation of therapy and before the start of the subsequent course. To investigate the mechanisms of action of BV in the bone marrow biopsies, microvessel density (MVD), cellular expression of VEGF, VEGFR2 and the activation status of VEGFR2 at tyrosine phosphorylation sites 951 and 996 (phosphorylated VEGFR2 [p-VEGFR-2] at Y951 and Y996) were analyzed by immunohistochemical staining as described.1,4 Before and one day after each BV infusion, we determined circulating molecular angiogenic factors in peripheral blood such as VEGF protein in citrated plasma, circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPCs) by fluorescence activated cell sorting as reported by others.5,6 Complete blood cell counts were examined twice a week. Safety was assessed at least weekly by physical examination, recording of vital signs and laboratory tests (hematology, coagulation and clinical chemistry). During treatment with BV, none of the patients fulfilled the criteria of a partial response, defined as the clearance of at least 50% marrow blasts accompanied by increases in platelet counts and hemoglobin values. In five patients with a pretreatment blast percentage of X90%, no change of blast counts at day 14 and day 35 marrow aspirates and biopsies were observed. One patient showed progressive disease with an increase from 50% blasts pretreatment and 95% blasts after the first cycle on day 14. Only one patient showed a reduction of marrow blasts (40% at presentation; 25% on day 14). Two patients were not

Change in molecular markers and blast cell counts in bone marrow and peripheral blood during BV treatment

Marker

VEGF-A VEGFR2 p-VEGFR2 (Tyr951) p-VEGFR2 (Tyr996) MVD KDR pos cells % in PB EPCs abs. in PB CECs abs. in PB VEGF plasma levels Blast cell % in BM

BL (before C1)

C2 (day 14 after C1)

C3 (day 35 after C1)

BL to C2/3

No. of patients

Median (range)

No. of patients

Median (range)

No. of patients

Median (range)

No. of patients

Pn

8 8 9 9 8 7 9 6 9 9

4.1 (1.5–5.8) 4.4 (3.3–13) 2.8 (1.6–8.3) 2.7 (1.1–10.3) 41 (25–76) 41 (7–95) 127 (3–497) 52 (9–148) ND 77 (40–90)

7 7 7 7 7 5 6 4 7 7

2.1 (1.3–5) 6 (5–10.9) 5.3 (1–7) 4.0 (1.0–5.1) 40 (26–58) 38 (3–86) 133 (7–256) 61 (20–158) ND 82 (25–95)

2 2 2 2 2 3 3 3 2 2

2 (1.5–2.5) 2.62 (2.2–3) 5.25 (4.6–5.9) 4.1 (1–5) 37 (30–44) 8 (4–12) 350 (18–1078) 28 (10–51) ND 90 (90)

7 7 7 7 7 5 6 4

0.042 0.398 0.735 0.612 0.499 0.500 0.116 0.465

n P-values were calculated using the Wilcoxon signed rank test. Abbreviations: BL, baseline; BM, bone marrow; C, cycle; CECs, circulating endothelial cells; EPCs, endothelial progenitor cells; MVD, microvessel density; ND, not detectable; PB, peripheral blood; p-VEGFR2, phosphorylated VEGFR2.

Leukemia

Letters to the Editor

1312

Figure 1 Immunohistochemical staining of the vascular endothelial growth factor expression in biopsy specimens of AML bone marrow during treatment with BV. (a) VEGF at baseline on left and post-cycle 1 on right. (b) Changes in VEGF expression in all patients before and after BV treatment. AU, arbitrary units (the proportion and intensity of stained cells scored in each biopsy).

evaluable after BV treatment due to progressive disease and death. Table 2 summarizes the median and range of bone marrow blast percentage in the nine patients before and after BV. Anti-VEGF therapy with BV did not have any significant effect on blast cell infiltration. In contrast, the level of VEGF expression in the bone marrow significantly decreased during treatment with BV as shown in Table 2 and Figure 1a and b (before BV treatment: median 3.78 (arbitrary units) AU, range 1.5–5.8 AU; after BV treatment: median 2.57 AU, range 1.3–5.0 AU; Wilcoxon test; P ¼ 0.042). However, there was no significant change in VEGFR2 and p-VEGFR2 (Y951 and Y996) expression in the bone marrow, suggesting that phosphorylation at the two different sites of VEGFR2 were not inhibited by BV (see Table 2). Furthermore, MVD was not influenced by BV administration, and neither plasma VEGF (measured by ELISA) nor cellular VEGFR2, CECs or CEPCs (measured by flow cytometry) changed significantly (see Table 2). Together, our data demonstrate that the antiangiogenic agent BV had a significant effect on the decrease in cellular VEGF expression in the bone marrow after 1–3 cycles BV without displaying any significant antileukemic activity in this heavily pretreated group and any significant effects on surrogate markers of the antiangiogenic response. However, BV was well tolerated with a favorable safety profile. In our cohort three patients suffered from pre-existing grade 2 hypertension, and their blood pressure was controlled with escalation of antihypertensive medication (calcium-channel blockers, angiotensin-converting-enzyme inhibitors and diuretics). There were no hypertensive crisis or deaths related to hypertension in this patient cohort. No venous or arterial thromboembolic events, and no bleeding despite low platelet counts or proteinuria was seen. Therefore, our data should not discourage the initiation of studies investigating the potential of combining antiangiogenic treatment strategies with conventional cytotoxic drug regimens. A pilot study exploring this

approach has shown promising CR rates in combination with cytarabine and mitoxantrone.7 Further studies are necessary to define its exact role.

L Zahiragic, C Schliemann, R Bieker, NH Thoennissen, K Burow, C Kramer, M Zu¨hlsdorf, WE Berdel and RM Mesters Department of Medicine/Haematology and Oncology, University of Muenster, Muenster, Germany E-mail: [email protected] References 1 Padro T, Bieker R, Ruiz S, Steins M, Retzlaff S, Bu¨rger H et al. Overexpression of vascular endothelial growth factor (VEGF) and its cellular receptor KDR (VEGFR-2) in the bone marrow of patients with acute myeloid leukemia. Leukemia 2002; 16: 1302–1310. 2 Gerber HP, Ferrera N. The role of VEGF in normal and neoplastic hematopoiesis. J Mol Med 2003; 57: 4593–4599. 3 Dias S, Hattori K, Heissig B, Zhu Z, Wu Y, Witte L et al. Inhibition of both paracrine and autocrine VEGF/VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias. Proc Natl Acad Sci USA 2001; 98: 10857–10862. 4 Mesters RM, Padro T, Bieker R, Steins M, Kreuter M, Goner M et al. Stable remission after administration of the receptor tyrosine kinase inhibitor SU5416 in a patient with refractory acute myeloid leukemia. Blood 2001; 98: 241–243. 5 Massa M, Rosti V, Ramajoli I, Campanelli R, Pecci A, Viarengo G et al. Circulating CD34+, CD133+, and vascular endothelial growth factor receptor 2-positive endothelial progenitor cells in myelofibrosis with myeloid metaplasia. J Clin Oncol 2005; 23: 5688–5695. 6 Zhang H, Vakil V, Braunstein M, Smith EL, Maroney J, Chen L et al. Circulating endothelial progenitor cells in multiple myeloma: implications and significance. Blood 2005; 105: 3286–3294, Epub 23 December 2004. 7 Karp JE, Gojo I, Pili R, Gocke CD, Greer J, Guo C et al. Targeting vascular endothelial growth factor for relapsed and refractory adult acute myelogenous leukemias: therapy with sequential 1-b-Darabinofuranosylcytosine, mitoxantrone, and bevacizumab. Clin Cancer Res 2004; 10: 3577–3585.

Angiopoietin-2 expression in B-cell chronic lymphocytic leukemia: association with clinical outcome and immunoglobulin heavy-chain mutational status

Leukemia (2007) 21, 1312–1315. doi:10.1038/sj.leu.2404650; published online 15 March 2007

For several years chronic lymphocytic leukemia (CLL) has been considered a disease of accumulation of relentless matureLeukemia

looking malignant monoclonal B cells due to a presumed defect in programmed cell death. However, several observations suggest that CLL cells are not immortal and that they require signals from the microenvironment to maintain viability. Increasing evidence shows that neovascularization plays a role in the biology of CLL.1 Increased microvessel density in marrow