Hematopathology / VEGF EXPRESSION IN ACUTE MYELOID LEUKEMIA Immunohistochemical Detection of VEGF in the Bone Marrow of Patients With Acute Myeloid Leukemia Correlation Between VEGF Expression and the FAB Category

We studied vascular endothelial growth factor (VEGF) expression in bone marrow sections obtained from 3 healthy donors and 41 patients with acute myeloid leukemia (AML) of various French-AmericanBritish (FAB) subtypes by immunohistochemical analysis using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody reacted with myeloid progenitor cells and megakaryocytes but not with erythroid cells or mature granulocytic cells. High levels of VEGF were found in the bone marrow in patients with AML-M1, -M2, -M3, -M4, -M4Eo, and -M5. In these leukemias, the vast majority of myeloblasts (>90%) expressed VEGF. By contrast, in AML-M0, the percentage of VEGF-positive blasts was lower in most cases (median, 42%), and if at all detectable, these blast cells contained only trace amounts of VEGF. In AML-M3 and -M4Eo, maturing granulocytes failed to express VEGF similar to granulocytes in normal bone marrow. In AML-M6, myeloblasts exhibited VEGF, whereas erythroid cells did not. In AML-M7, blast cells and megakaryocytes were identified as major sources of VEGF. In summary, VEGF expression in the bone marrow is restricted to certain stages of differentiation and maturation of myeloid cells and correlates with the FAB category. Acute myeloid leukemia (AML) is characterized by clonal proliferation of immature myeloid (progenitor) cells without substantial maturation.1,2 The prognosis and clinical picture in AML varies depending on the genes that underwent deregulation, cell types involved, and the specific biologic properties of the clone.3-7 Traditionally, the AMLs have been classified according to the proposal of the FrenchAmerican-British (FAB) Cooperative Study Group.8-11 More recently, this proposal has been extended and modified by guidelines provided in the World Health Organization classification of tumors of hematopoietic and lymphoid tissues.12,13 Recent data suggest that vascular endothelial growth factor (VEGF) is overexpressed in patients with AML and has a potential role in the pathogenesis of myeloid leukemias.14-19 In fact, VEGF has been implicated as an essential mediator of leukemia-dependent angiogenesis.14-19 In addition, this cytokine has been described as an autocrine and paracrine growth regulator of AML cells.18-20 Indeed, VEGF is produced and secreted in AML blasts in a constitutive manner.14-19 Correspondingly, increased levels of VEGF are detectable in serum samples of patients with AML.14-19 However, little is known about VEGF expression in various subtypes of AML and about the exact distribution of this cytokine in the cellular compartments of the normal and leukemic bone marrow. Recently, a VEGF-staining protocol suitable for detection of the cytokine in paraffin-embedded bone marrow biopsy sections has been established.21 In the present study, we applied this protocol to a cohort of 41 patients with AML to study expression and distribution of VEGF in the bone marrow in various subtypes of AML. Am J Clin Pathol 2003;119:663-671 663 663 DOI: 10.1309/331QX7AXKWFJFKXM 663 © American Society for Clinical Pathology Ghannadan et al / VEGF EXPRESSION IN ACUTE MYELOID LEUKEMIA Materials and Methods Patients A total of 41 patients (18 females; 23 males; F/M ratio, 1:1.3) with AML were examined. The median age was 63 years (range, 2-86 years). Bone marrow was obtained from the iliac crest after informed consent was given. Diagnoses were established using FAB criteria.8-11 Accordingly, patients had the following subtypes of AML: M0, 6; M1, 5; M2, 5; M3, 4; M4, 5; M4Eo, 4; M5, 5; M6, 5; and M7, 2. Patient characteristics are given in ❚Table 1❚. For control purposes, we also examined 3 patients with normal bone marrow (staging in nonHodgkin lymphomas). Immunohistochemical Analysis Immunohistochemical analysis was performed on paraffin-embedded, formalin-fixed bone marrow sections using the indirect immunoperoxidase staining technique as described.21 Endogenous peroxidase was blocked by methanol–hydrogen peroxide. Before staining with a polyclonal rabbit anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA), sections were pretreated by microwave oven. The anti-VEGF antibody (work dilution, 1:50) was diluted in a 0.05-mol/L concentration of tris-buffered saline (pH 7.5) plus 1% bovine serum albumin, and applied for 60 minutes. In selected experiments, anti-VEGF was preincubated with a specific (VEGF-related) blocking-peptide (Santa Cruz Biotechnology). After washing, slides were incubated 664 Am J Clin Pathol 2003;119:663-671 664 DOI: 10.1309/331QX7AXKWFJFKXM © American Society for Clinical Pathology ❚Table 1❚ Patient Characteristics Case No./Sex/Age FAB Subtype Blasts in Bone WBC Count, /μL Hemoglobin, g/dL Platelet Count, × 103/μL at Diagnosis (y) of AML Marrow Smears (%) (× 109/L) (g/L) (× 109/L) 1/F/50 M0 94 26,500 (26.5) 7.8 (78) 10 (10) 2/M/70 M0 85 13,900 (13.9) 9.9 (99) 71 (71) 3/M/33 M0 91 5,200 (5.2) 8.6 (86) 51 (51) 4/M/65 M0 34 1,700 (1.7) 8.6 (86) 24 (24) 5/M/68 M0 70 2,600 (2.6) 11.9 (119) 215 (215) 6/M/31 M0 30 2,100 (2.1) 9.9 (99) 33 (33) 7/F/26 M1 82 3,700 (3.7) 6.2 (62) 55 (55) 8/M/79 M1 87 101,200 (101.2) 8.9 (89) 26 (26) 9/F/73 M1 93 19,200 (19.2) 10.9 (109) 52 (52) 10/M/71 M1 87 75,200 (75.2) 14.4 (144) 61 (61) 11/F/52 M1 91 9,700 (9.7) 10.2 (102) 71 (71) 12/M/65 M2 60 65,200 (65.2) 7.2 (72) 93 (93) 13/F/80 M2 36 1,100 (1.1) 9.7 (97) 145 (145) 14/M/63 M2 48 5,000 (5.0) 9.5 (95) 56 (56) 15/M/47 M2 60 12,600 (12.6) 8.7 (87) 56 (56) 16/F/41 M2 75 6,000 (6.0) 10.8 (108) 83 (83) 17/M/32 M3 90 120,000 (120.0) 12.3 (123) 20 (20) 18/F/15 M3 90 600 (0.6) 5.2 (52) 6 (6) 19/M/44 M3 85 24,800 (24.8) 11.1 (111) 11 (11) 20/M/6 M3 83 8,000 (8.0) 6.9 (69) 32 (32) 21/F/70 M4 75 27,200 (27.2) 9.1 (91) 48 (48) 22/M/78 M4 36 222,400 (222.4) 8.9 (89) 39 (39) 23/F/70 M4 65 9,500 (9.5) 7.8 (78) 26 (26) 24/F/53 M4 80 300,000 (300.0) 6.6 (66) 54 (54) 25/M/70 M4 38 19,900 (19.9) 8.3 (83) 215 (215) 26/F/25 M4Eo 38 17,400 (17.4) 9.3 (93) 15 (15) 27/F/64 M4Eo 34 29,000 (29.0) 9.3 (93) 51 (51) 28/F/38 M4Eo 49 2,800 (2.8) 8.2 (82) 66 (66) 29/M/77 M4Eo 33 5,600 (5.6) 8.8 (88) 48 (48) 30/M/75 M5 72 12,500 (12.5) 7.5 (75) 5 (5) 31/F/61 M5 80 61,800 (61.8) 10.8 (108) 104 (104) 32/F/34 M5 85 102,000 (102.0) 9.9 (99) 138 (138) 33/F/21 M5 84 190,000 (190.0) 11.0 (110) 51 (51) 34/M/66 M5 92 37,500 (37.5) 9.2 (92) 20 (20) 35/M/58 M6 25 2,600 (2.6) 9.3 (93) 70 (70) 36/M/75 M6 8 3,700 (3.7) 8.2 (82) 25 (25) 37/M/86 M6 10 2,200 (2.2) 12.8 (128) 25 (25) 38/M/38 M6 25 1,800 (1.8) 7.7 (77) 30 (30) 39/F/63 M6 9 1,900 (1.9) 11.6 (116) 131 (131) 40/F/70 M7 40 2,000 (2.0) 9.5 (95) 22 (22) 41/M/2 M7 30 4,700 (4.7) 7.4 (74) 15 (15) AML, acute myeloid leukemia; FAB, French-American-British Cooperative Study Group classification.